Covalently cross-linked oligonucleotides

ABSTRACT

Covalent cross-linkages for two oligonucleotide strands or for first and second regions of a single oligonucleotide strand connect sugar moieties of nucleotides on the respective strands or the regions of the single strand. The cross-linkages are connected to at least one strand or region via a space-spanning group. The cross-linkage also can be connected to the other strand or other region via a space-spanning group or via an abasic site located on the other strand or other region.

This is the national stage filing of application PCT/US93/02059, filed 5 Mar. 1993, which is a continuation-in-part of application Ser. No. 07/846,376, filed 5 Mar. 1992, now abandoned.

FIELD OF THE INVENTION

This invention is directed to covalent cross-linkages for oligonucleotides. The cross-linkages connect either two oligonucleotide strands or different regions of a single oligonucleotide strand via a space-spanning group that extends from a sugar moiety on a nucleotide of the first strand or strand region to a nucleotide on the other strand or strand region.

BACKGROUND OF THE INVENTION

Several examples have been reported of cross-linking between complementary oligonucleotides. The purpose of such cross-linking often has been to disrupt the function of a nucleic acid, primarily by cleavage of the target. It is known to cross-link nucleic acids in a positionally uncontrolled manner with UV exposure. However, Grineva, and Karpova, FEBS., 1973, 32, 351-355, appear to have been the first to covalently cross-link complementary strands of oligonucleotides at a specific site utilizing controlled chemistry. A nitrogen mustard was attached to the 3' terminal ribose unit of an oligonucleotide via an acetal linkage or to the 5' end of an oligonucleotide via a phosphoramide linkage. On hybridization, the reactive mustards covalently cross-linked to the complementary strand via alkylation of the ternary heteroaromatic nitrogen atom at the 7-position of guanine or adenine. However, the alkylated base thus formed is a quaternary charged species that is subject to rapid chemical degradation via imidazole ring opening followed by cleavage of the targeted strand.

Enzymes are known to remove alkylated bases from nucleic acids. Indeed, such enzymatic removal is implicated in DNA repair mechanisms. The destruction of a targeted alkylated nucleic acid theoretically could be utilized for antimicrobial or antitumor therapy. However, cross-linking alkylation reactions and subsequent degradations are not desirable where host cell viability is necessary.

Summerton and Bartlett have proposed another type of alkylated oligonucleotide wherein an α-bromomethylketone attached to the 4-position of a cytidine nucleotide spans the major groove and alkylates the 7-position of a complementary guanine residue in a targeted strand. See, J. Mol. Biol., 1978, 122, 145-162; J. Theor. Biology, 1979, 78, 61-75; and U.S. Pat. No. 4,123,610!. As with the nitrogen mustards noted above, this alkylation yields a charged species that effects cleavage of the targeted nucleic acid strand following alkylation.

A further example of a covalent cross-linkage to the 7-position nitrogen of guanine is described by Meyer et. al., J. Am. Chem. Soc., 1989, 111, 8517. The authors attached an iodoacetamidopropyl moiety to the 5-position of a thymidine nucleotide of DNA. The iodoacetamidopropyl moiety subsequently alkylated the 7-position of a guanine nucleotide at a position two base pairs down the complementary strand. Cleavage of the targeted strand was observed at various times and temperatures.

Matteucci and Webb have described a hybridization. triggered cross-linking process where an N6,N6-ethano-adenine or N4,N4-ethanocytosine alkylates an appropriately positioned nucleophile in a complementary strand. See, Nucleic Acids Res., 1986, 14, 7661; Tetrahedron Letters, 1987, 28, 2469-2472.! This process has been designed to inactivate the normal function of the targeted DNA either by forming a stable adduct or by hydrolytic or enzymatic cleavage.

A cross-linkage similar to the above-noted N6,N6-ethano linkage was described by Ferentz, et al., J. Am. Chem. Soc., 1991, 113, 4000, who utilized either an ethyl disulfide or a propyl disulfide linkage to connect the N6-positions of adenine residues on a self-complementary oligonucleotide.

Lee, et al., Biochemistry, 1988, 27, 3197-3203, described the interaction of psoralen-derivatized methyl phosphonate oligonucleotides with single-stranded DNA. According to this method, irradiation effects cross-linking which, in turn, inactivates DNA. Psoralen cross-linkages occur only between pyrimidine bases to join single-stranded or, more commonly, double-stranded structures.

The generation of an abasic site on a nucleic acid has been implicated in the above-noted enzymatic removal of alkylated bases from nucleic acids. Manoharan, et al., J. Am. Chem. Soc., 1987, 109, 7217 and Manoharan, et al., J. Am. Chem. Soc., 1988, 110, 2690, have characterized abasic sites in oligonucleotides utilizing ¹³ C NMR spectroscopy. Abasic sites also have been created on nucleic acids to attach the intercalator 9-aminoellipticine to nucleic acids. Vasseur, et al., Nucleosides & Nucleotides, 1989, 8, 863-866, reported that the trimer Tp(Ap)Pt, where (Ap) is an apurinic site yielded a mixture of products when reacted with the intercalator 9-aminoellipticine. The reaction produced a desired Schiff's base adduct between the apurinic site and the 9-aminoellipticine, but also resulted in cleavage of the phosphate backbone. This work was extended by Bertrand, et al., Nucleic Acids Research, 1989, 17, 10307, to the interaction of 9-aminoellipticine and a 13-mer oligonucleotide. The 13-mer oligonucleotide contained only a single purine nucleotide. Acid treatment resulted in loss of the purine base to yield an unstable abasic site. The 9-aminoellipticine was successfully linked to the abasic site in the oligonucleotide by reducing the Schiff's base adduct during the reaction. Using an oligonucleotide having a single purine site, the authors were able to study the coupling of the intercalator 9-aminoellipticine to the oligonucleotide. It is unlikely, however, that oligonucleotides which bind to sequences of biological significance contain only a single purinic site.

Other abasic site-containing oligonucleotides have been assembled by synthetically incorporating abasic nucleoside precursors into an oligonucleotide. For example, Philippe, et al., Tetrahedron Letters, 1990, 31, 6347-6350, incorporated a 2-deoxy-D-ribose having a primary amide function attached via a pentamethylene chain to its 1'-position. 1,2-Dideoxy-D-ribofuranose and butane 1,3-diol likewise have been incorporated into oligonucleotides. See, P. lyer, et al., Nucleic Acids Research, 1990, 18, 2855.!

Groehke, et al., Helvetica Chimica Acta, 1990, 73, 608, reported the preparation of a (tert-butyl)dimethylsilyl-protected deoxy-D-ribose and its incorporation into oligonucleotides utilizing solid state techniques. Reoc'h, et al., Tetrahedron Letters, 1991, 32, 207, used 1-(o-nitrobenzyl)-2-deoxy-D-ribofuranose in the synthesis of other oligonucleotides. None of these chemically generated abasic sites have been utilized to effect cross-linking.

Application PCT/US91/01822, filed Mar. 19, 1991 and entitled Reagents And Methods For Modulating Gene Expression Through RNA Mimicry, discloses effective use of oligonucleotides that mimic RNA structure. The entire disclosure Application PCT/US91/01822, which is assigned to the assignee of this application, is herein incorporated by reference. The oligonucleotides disclosed in PCT/US91/01822 are selected to mimic a portion of an RNA encoded by a gene. The RNA-mimicking oligonucleotides interfere with or modulate gene expression by interfering with protein-RNA interactions.

In order to mimic an RNA, an oligonucleotide must assume and retain the RNA's secondary and tertiary structure. Although the physical and chemical forces that normally retain the RNA in its secondary and tertiary structure are non-covalent in nature, one way to fix an RNA mimic in the necessary secondary and tertiary structure would be to covalently cross-link the mimic. However, presently-known techniques for cross-linking nucleic acid strands either lead to strand cleavage, disruption of structure or Watson/Crick hydrogen bonding, or are only useful for limited, specific sequences or at specific locations in a sequence.

Cross-linkages between two oligonucleotide strands or between regions of a single strand would be undesirable where the cross-linkages could destroy the strands or strand or could unduly modify the conformational structure of the oligonucleotide. Cross-linkages also would be undesirable where they do not allow the cross-linked product to approximate the conformation of natural nucleic acids. Heretofore, there has be no suggestions in the art of cross-linkages or methods of cross-linking that do not destroy the strands, that allow suitable conformations, that are useful on various sequences and at various positions within the sequences, and that allow normal ranges of features such as the "tilt" and "propeller twist" features found in naturally occurring nucleic acid duplexes. Accordingly there remains a long-felt need for nucleic acid cross-linkages and methods of cross-linking nucleic acids.

OBJECTS OF THE INVENTION

It is a primary object of this invention to provide improved covalently cross-linked nucleic acids and methods of forming the same.

It is a further object of this invention to provide individual oligonucleotide strands that are covalently bonded to one another at one or more sites on the strands.

It is an even further object of this invention to provide a single oligonucleotide strand that is covalently bonded to itself at one or more sites on the strand.

It is an even further object of this invention to provide RNA mimics that are fixed in specific spatial conformations via cross-linking covalent bonds.

It is an additional object of this invention to provide covalently cross-linked oligonucleotides that have the same sequence as known nuclease-resistant mimics of binding receptors for nucleic acid-binding proteins.

These and other objects of this invention will become apparent to those skilled in the art from a review of this specification and the claims appended hereto.

BRIEF DESCRIPTION OF THE INVENTION

In accordance with this invention there are provided sequence-specific, covalently cross-linked nucleic acids comprising a first nucleotide located either on a first strand of complementary oligonucleotide strands or on a single oligonucleotide strand. The cross-linked nucleic acids further comprise a second nucleotide located on a further strand of the complementary strands or on the single strand at a site distal to the first nucleotide. A first bond means is located on a sugar moiety of the first nucleotide and a second bond means is located on a sugar moiety of the second nucleotide. A covalent cross-linkage connects the first and the second bond means and, in doing so, cross-links the strand or strands.

In one embodiment of the invention, the first and the second nucleotides are located within first and second nucleotide sequences, respectively, on a single oligonucleotide strand. In a first preferred embodiment, the second nucleotide sequence is separated from the first nucleotide sequence by a distance that allows the oligonucleotide strand to assume a conformation wherein the first and second nucleotide sequences are specifically hybridizable with one another. In a further preferred embodiment, the first and the second nucleotides are located in non-hybridizable positions but are in sufficient proximity to allow cross-linking between the first and second nucleotides.

In a preferred embodiment of the invention, the first bond means includes an abasic site and the second bond means includes a space-spanning group that, in turn, includes an active functional group that is capable of covalently bonding with the abasic site.

Further in accordance with the invention there are provided sequence-specific, cross-linked oligonucleotides that include a first nucleotide located on a first complementary oligonucleotide strand, a second nucleotide located on a second complementary strand, a first bond precursor located on a sugar moiety of the first nucleotide, a second bond precursor located on a sugar moiety of the second nucleotide, and a covalent cross-linkage connecting the first and the second bond precursors to cross-link the first and the second strands. In a preferred embodiment of the invention, the first nucleotide is located in a first nucleotide sequence, the second nucleotide is located in a second nucleotide sequence, and the second nucleotide sequence is complementary to and specifically hybridizable with the first nucleotide sequence.

Additionally in accordance with this invention there are provided sequence-specific, cross-linked oligonucleotides that include a first nucleotide located on an oligonucleotide strand and a second nucleotide located on the same oligonucleotide strand. The first and second nucleotides are separated by a distance that allows the nucleotides to specifically hybridize with one another. A first bond precursor is located on a sugar moiety of the first nucleotide and a second bond precursor is located on a sugar moiety of the second nucleotide. A covalent cross-linkage connects the first and the second bond precursors, cross-linking the first and second nucleotides. In a preferred embodiment, the first nucleotide is located in a first nucleotide sequence, the second specific nucleotide is located in a second nucleotide sequence, and the second nucleotide sequence is complementary to and hybridizable with the first nucleotide sequence. In a further preferred embodiment, the first and second nucleotides are located in nonhybridizable positions but are in sufficient proximity that a cross-linkage can be formed between them.

Also in accordance with this invention there are provided cross-linked oligonucleotides comprising a first sequence region that includes an abasic site and a second sequence region that includes a space-spanning group. An active functional group is located on the space-spanning group and is covalently coupled with the abasic site. In a first preferred embodiment, the first sequence region and the second sequence region are on the same oligonucleotide strand. In a further preferred embodiment, the first sequence region and the second sequence region are on different oligonucleotide strands. In these preferred embodiments, the first sequence region and the second sequence region are complementary to and specifically hybridizable with one another. Preferably, the active functional group is an amine, hydrazine, hydroxylamine, semicarbazide, thiosemicarbazide, hydrazide, thiol or alcohol, and the abasic site includes an aldehydic functionality.

Space-spanning groups according to the invention comprise elongated chains from about 1 to about 50 atoms in length. Space-spanning groups preferably comprise elongated chains from about 4 to about 20 atoms in length, more preferably chains from about 8 to about 13 atoms in length.

Further in accordance with this invention there are provided cross-linked oligonucleotides that include a first sequence region, a second sequence region, and a covalently bonded linker of the structure:

    X.sub.1 --L.sub.1 --Y--L.sub.2 --X.sub.2

wherein X₁ is covalently connected to the first sequence region and X₂ is covalently connected to the second sequence region and where:

(i) X₁ and X₂, independently, are O, S, NH, CH₂ or CO;

L₁ and L₂, independently, are C₁ -C₂₀ straight chain alkyl, C₁ -C₂₀ straight chain substituted alkyl, C₂ -C₅₀ branched chain alkyl, C₂ -C₅₀ branched chain substituted alkyl, C₂ -C₂₀ straight chain alkenyl, C₂ -C₂₀ straight chain substituted alkenyl, C₃ -C₅₀ branched chain alkenyl, C₃ -C₅₀ branched chain substituted alkenyl, C₂ -C₂₀ straight chain alkyne, C₂ -C₂₀ straight chain substituted alkyne, C₃ -C₅₀ branched chain alkyne, C₃ -C₅₀ branched chain substituted alkyne, polyamine, polyamide, polyester, polyethylene glycol, polyether, aryl, aralkyl or heterocyclic; and

Y is an imine, amine, oxime, hydroxylamine, hydrazine, hydrazone, azine, hydrazide-hydrazone, amide, hydrazide, semicarbazide, semicarbazone, thiosemicarbazide, thiocarbazone, disulfide, hemiacetal, thiohemiacetal, α-keto-alkylthioalkyl or α-keto-alkylaminoalkyl; or

(ii) X₁ and X₂, independently, are O or NH; and

L₁ --Y--L₂ together are NH--L₃ --NH or NH--NH--L₃ --NH--NH; and

L₃ is C₁ -C₂₀ straight chain alkyl, C₁ -C₂₀ straight chain substituted alkyl, C₂ -C₅₀ branched chain alkyl, C₂ -C₅₀ branched chain substituted alkyl, C₂ -C₂₀ straight chain alkenyl, C₂ -C₂₀ straight chain substituted alkenyl, C₃ -C₅₀ branched chain alkenyl, C₃ -C₅₀ branched chain substituted alkenyl, C₂ -C₂₀ straight chain alkyne, C₂ -C₂₀ straight chain substituted alkyne, C₃ -C₅₀ branched chain alkyne, C₃ -C₅₀ branched chain substituted alkyne, polyamine, polyamide, polyester, polyethylene glycol, polyether, aryl, aralkyl or heterocyclic; or

(iii) X₁ and X₂, independently, are O, S, NH, CH₂ or CO;

L₁ and L₂, independently, are C₁ -C₂₀ straight chain alkylamino, C₁ -C₂₀ straight chain substituted alkylamino, C₂ -C₅₀ branched chain alkylamino, C₂ -C₅₀ branched chain substituted alkylamino, C₂ -C₂₀ straight chain alkenylamino, C₂ -C₂₀ straight chain substituted alkenylamino, C₃ -C₅₀ branched chain alkenylamino, C₃ -C₅₀ branched chain substituted alkenylamino, C₂ -C₂₀ straight chain alkynylamino, C₂ -C₂₀ straight chain substituted alkynylamino, C₃ -C₅₀ branched chain alkynylamino, C₃ -C₅₀ branched chain substituted alkynylamino, C₁ -C₂₀ straight chain alkylthio, C₁ -C₂₀ straight chain substituted alkylthio, C₂ -C₅₀ branched chain alkylthio, C₂ -C₅₀ branched chain substituted alkylthio, C₂ -C₂₀ straight chain alkenylthio, C₂ -C₂₀ straight chain substituted alkenylthio, C₃ -C₅₀ branched chain alkenylthio, C₃ -C₅₀ branched chain substituted alkenylthio, C₃ -C₂₀ straight chain alkynylthio, C₂ -C₂₀ straight chain substituted alkynylthio, C₃ -C₅₀ branched chain alkynylthio or C₃ -C₅₀ branched chain substituted alkynylthio; and

Y is a heterobifunctional or homobifunctional cross-linking reagent;

where said substituent of said substituted alkyl, substituted alkenyl and substituted alkyne moieties is an intercalator, a conjugate, polyamine, polyamide, polyethylene glycol, a group that enhances the pharmacodynamic properties of oligonucleotides or a group that enhances the pharmacokinetic properties of oligonucleotides.

Further in accordance with this invention there are provided cross-linked oligonucleotides that individually include a first sequence region, a second sequence region, a first space-spanning group connected to a 2'-position of a nucleotide located in the first sequence region, a second space-spanning group connected to a 2'-position of a nucleotide in the second sequence region, and a linking moiety covalently connecting the first and second space-spanning groups. In preferred embodiments the linking moiety is:

--N═CH--, --N(R)--, --N(R)--CH₂ --N(R)--, --O --N═CH--, --O--N(R)--, --N(R)--N(R)--, --N(R)--N═CH--, --HC═N--N═CH--, --C(O)--N(R)--N═CH--, --C(O)N(R)--, --C(O)--N(R)--N(R)--, --N(R)--C(O)--N(R)--N(R)--, --N(R)--C(O)--N(R)--N(R)═CH--, --N(R)--C(S)--N(R)--N(R)--, --N(R)--C(O)--N(R)--N(R)═CH--, --CH(OH)--O--CH₂ --, --CH (OH)--S --CH₂ --, --CH₂ --S--CH₂ --C(O)--CH₂ --, --CH₂ --NH--CH₂ --C(O)--CH₂ -- or --S--S--;

wherein R is H, alkyl, alkenyl, aralkyl, an intercalator, a conjugate, polyamine, polyamide, polyethylene glycol, a group that enhances the pharmacodynamic properties of oligonucleotides or a group that enhances the pharmacokinetic properties of oligonucleotides.

Further in accordance with this invention there are provided methods for fixing a sequence-specific oligonucleotide in a set configuration. Such methods include selecting a first site in the oligonucleotide, said first site having a first nucleotide sequence. The methods further include selecting a second site in the oligonucleotide, said second site having a second nucleotide sequence. A first bond precursor is attached to a sugar moiety of a first nucleotide of the first site, while a second bond precursor is attached to a sugar moiety of a second nucleotide of the second site. A covalent cross-linkage is formed between the first and the second bond precursors. In a preferred embodiment, the first bond precursor is attached to the first site by reacting a first nucleotide precursor with a reagent for introducing the first bond precursor on a sugar moiety of the first nucleotide precursor. The first nucleotide precursor is derivatized to a protected and activated form and is synthetically incorporated into the first specific nucleotide sequence. The second nucleotide sequence can be complementary to and hybridizable with the first nucleotide sequence.

In one embodiment of this method, the first bond precursor is an abasic nucleotide and the second bond precursor is attached to a 2'-position of the second nucleotide. In this embodiment, the second bond precursor is reacted with the abasic site to form an imine, oxime, hydrazone, hydrazide-hydrazone, semicarbazone, thiosemicarbazone, hemiacetal or thiohemiacetal linkage between the first nucleotide sequence and the second nucleotide sequence. Certain of these linkages can be reduced to amine, hydroxylamine, hydrazine, hydrazide, semicarbazide or thiosemicarbazide linkages.

In a further embodiment of the method, the first bond precursor is attached to a 2'-position of the first nucleotide and the second bond precursor is attached to a 2'-position of the second nucleotide. Preferred linkages for this embodiment include:

--N═CH--, --N(R)--, --N(R)--CH₂ --N(R)--, --O--N═CH--, --O--N(R)--, --N(R)--N(R)--, --N(R)--N═CH--, --HC═N--N═CH--, --C(O)--N(R)--N═CH--, --C(O)N(R)--, --C(O)--N(R)--N(R)--, --N(R)--C(O)--N(R)--N(R)--, --N(R)--C(O)--N(R)--N(R)═CH--, --N(R)--C(S)--N(R)--N(R)--, --N(R)--C(S)--N(R)--N(R)═CH--, --CH(OH)--O--CH₂ --, --CH(OH)--S--CH₂ --, --CH₂ --S--CH₂ --C(O)--CH₂ --, --CH₂ --NH--CH₂ --C(O)--CH₂ -- or --S--S--;

wherein R is H, alkyl, alkenyl, aralkyl, an intercalator, a conjugate, polyamine, polyamide, polyethylene glycol, a group that enhances the pharmacodynamic properties of oligonucleotides or a group that enhances the pharmacokinetic properties of oligonucleotides.

Further in accordance with this invention there are provided methods for cross-linking a single sequence-specific oligonucleotide strand. These methods include selecting first and second nucleotide sequences in the oligonucleotide, incorporating an abasic nucleotide within the first nucleotide sequence, attaching a bond precursor to one of the nucleotides of the second sequence, and forming a covalent cross-linkage between the abasic site and the bond precursor. In one embodiment of this method, the bond precursor is attached to a sugar moiety of the nucleotide of the second sequence, preferably at the 2'-position. In preferred embodiments, the abasic site includes an aldehydic functional group and the bond precursor includes a functional group capable of forming an oxime, imine, hydrazone, hydrazide-hydrazone, semicarbazone, thiosemicarbazone, hemiacetal or thiohemiacetal linkage with the aldehydic functional group. The second nucleotide sequence preferably is selected to be complementary to and specifically hybridizable with the first nucleotide sequence.

Further methods for cross-linking a single, sequence-specific oligonucleotide include selecting a first nucleotide sequence and a second nucleotide sequence in the oligonucleotide. A first bond precursor is attached to a sugar moiety of a nucleotide of the first nucleotide sequence and a second bond precursor is attached to a sugar moiety of a nucleotide of the second nucleotide sequence. A covalent cross-linkage is formed between the first and the second precursors. In preferred embodiments, the cross-linkage is an imine, amine, aminoalkylamine, oxime, hydroxylamine, hydrazine, hydrazone, azine, hydrazide-hydrazone, amide, hydrazide, semicarbazide, semicarbazone, thiosemicarbazide, thiocarbazone, disulfide, hemiacetal, thiohemiacetal, α-keto-alkylthioalkyl or α-keto-alkylaminoalkyl cross-linkage. In other preferred embodiments the second nucleotide sequence is complementary to and hybridizable with the first nucleotide sequence.

Further in accordance with this invention there are provided methods for cross-linking first and second strands of oligonucleotides, comprising the steps of selecting a first nucleotide sequence in a first oligonucleotide strand and selecting a second nucleotide sequence in a second oligonucleotide strand. The methods further include incorporating an abasic nucleotide within the first nucleotide sequence, attaching a bond precursor to a nucleotide of the second sequence, and forming a covalent cross-linkage between the abasic site and the bond precursor. In preferred embodiments, the second nucleotide sequence is complementary to and hybridizable with the first nucleotide sequence.

Further, in accordance with this invention there are provided methods for cross-linking first and second strands of oligonucleotides comprising the steps of selecting a first nucleotide sequence in a first oligonucleotide strand, selecting a second nucleotide sequence in a second oligonucleotide strand, attaching a first bond precursor to a sugar moiety of a nucleotide of the first sequence, locating a second covalent precursor on a sugar moiety of a nucleotide of the second sequence, and forming a covalent cross-linkage between the first and the second bond precursors. In preferred embodiments, the second nucleotide sequence is complementary to and hybridizable with the first nucleotide sequence.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the objects of this invention novel, covalently cross-linked oligonucleotide strands are disclosed. Preferably such strands are "duplexed" or "complementary" along at least a portion of their length. In the context of this invention the terminology "duplex strands" or "complementary strands" or like terminology refers in a first instance to two separate strands wherein at least one nucleotide or nucleotide sequence on one of the strands is complementary to and specifically hybridizable with an opposing nucleotide or nucleotide sequence on the other strand. In a second instance, this terminology refers to two sections or sequence regions of a single strand that are spaced from one another by a distance sufficient for the strand to fold onto itself and permit hybridization of complementary nucleotides in the respective sequence regions. Folded, self-hybridizing, single strands form hairpin loop, stem loop, interior loop or bulge type structures as a result of such hybridization.

Further in the context of this invention, the term "oligonucleotide" refers to polynucleotides formed from a plurality of joined nucleotide units. In certain instances, the nucleotides are formed from naturally occurring bases and pentofuranosyl sugars groups and are joined together via native inter-nucleoside phosphodiester linkages. The term "oligonucleotide" thus effectively includes naturally occurring or synthetic species formed from naturally occurring subunits. The term "oligonucleotide" also includes structures formed from modified or non-naturally occurring nucleotide subunits. Modifications can occur on the base portion of a nucleotide, on a sugar portion,of a nucleotide or on the linkage joining one nucleotide to the next. In addition, modifications can be made wherein nucleoside units are joined together with connecting groups that substitute for the inter-nucleoside phosphate linkages. Inter-nucleoside connecting groups, which are to be distinguished from the cross-linkages of the invention, include --O--CH₂ --CH₂ --O-- linkages and other novel linkages disclosed in U.S. Pat. No. 5,223,678, issued Jun. 29, 1993, and U.S. Pat. No. 5,378,825, issued Jan. 3, 1995, both assigned to the assignee of this invention. Other modifications can be made to the sugar, to the base, or to the phosphate group of the nucleotide. Exemplary modifications are disclosed in the following U.S. patent applications: Ser. No. 463,358, filed Jan. 11, 1990, entitled Compositions And Methods For Detecting And Modulating RNA Activity, now abandoned; Ser. No. 566,977, filed Aug. 13, 1990, entitled Sugar Modified Oligonucleotides That Detect And Modulate Gene Expression, now abandoned; U.S. Pat. No. 5,138,045, issued Aug. 17, 1992, Serial No. 558,806, filed Jul. 27, 1991, entitled Nuclease Resistant Pyrimidine Modified Oligonucleotides That Detect And Modulate Gene Expression and serial number PCT/US91/00243, filed Jan. 11, 1991, published Jul. 25, 1991, as WO 91/10671 and entitled Compositions and Methods For Detecting And Modulating RNA Activity, all assigned to the assignee of this invention. The disclosures of all of the above-noted patent applications are herein incorporated by reference.

The term oligonucleotides, as used in connection with this invention, refers to structures including modified portions such as modified sugar moieties, modified base moieties or modified sugar linking moieties. These modified portions function in a manner similar to natural bases, natural sugars and natural phosphodiester linkages. Thus, in the context of this invention, oligonucleotides may have altered base moieties, altered sugar moieties or altered inter-sugar linkages. Exemplary among these are: phosphorothioate, phosphorodithioate, methyl phosphonate, phosphotriester or phosphoramidate inter-nucleoside linkages used in place of phosphodiester inter-nucleoside linkages; deaza or aza purines and pyrimidines used in place of natural purine and pyrimidine bases; pyrimidine bases having substituent groups at the 5- or 6-positions; purine bases having altered or replacement substituent groups at the 2-, 6- or 8- positions; or sugars having substituent groups at their 2'-position, substitutions for one or more of the hydrogen atoms of the sugar, or carbocyclic or acyclic sugars. Oligonucleotides may also comprise other modifications consistent with the spirit of this invention. Such oligonucleotides are best described as being functionally interchangeable with yet structurally distinct from natural oligonucleotides. All such oligonucleotides are comprehended by this invention so long as they effectively mimic the function of a desired RNA or DNA strand.

Further in the context of this invention, it will be recognized that the terms "cross-linkage," "cross-link" or "cross-linking" specifically exclude those portions of the oligonucleotide structure that linearly connect individual nucleoside subunits or nucleoside-like, modified subunits, i.e., those portions that connect the nucleoside units in the sequence that defines the primary structure of the oligonucleotide. Thus, "cross-linkage," "cross-link" or "cross-linking" exclude native phosphodiester linkages that connect nucleosides, as well as analogous, non-native inter-nucleoside linkages such as phosphorothioate, phosphorodithioate methyl phosphonate, phosphotriester, phosphoramidate., and --O--CH₂ --CH₂ --O-- linkages used in place of phosphodiester inter-nucleoside linkages. The terms "cross-linkage," "cross-link" or "cross-linking" as used in the context of this invention refer to "additional" linkages created across a single oligonucleotide strand or several oligonucleotide strands to hold the strand or strands in secondary or other, higher-ordered structures. Primary, secondary, and higher-order structures are as defined in standard reference texts such as Nucleic Acids In Chemistry And Biology, edited by G. Michael Blackburn and Michael J. Gait, Oxford University Press, New York, 1991.

Cross-linking is employed in accordance with the present invention to fix separate oligonucleotide strands in duplex structures or to fix a single oligonucleotide strand in a hairpin loop, stem loop, interior loop, bulge or other similar higher-order structure. Such fixation is believed to confer nuclease resistance to oligonucleotides and to optimize structure-dependent function. Fixing a strand or strands in a duplex structure also can disrupt the normal function of single-strand nucleic acid-binding proteins by forming nuclease-resistant mimics of the proteins' binding receptors. Fixing a strand or strands in set secondary structures also makes it possible to mimic the RNA secondary structures found in diseased cells, particularly cells infected with viruses and retroviruses. A detailed delineation of such RNA mimicry is disclosed in Application PCT/US91/01822, filed Mar. 19, 1991, entitled Reagents and Methods For Modulating Gene Expression Through RNA Mimicry, assigned to the same assignee as this application. The entire contents of Application PCT/US91/01822 is herein incorporated by reference.

The "fixing" of multiple oligonucleotide strands or a single oligonucleotide strand in defined secondary or other higher-order structure is achieved in accordance with this invention by cross-linking the oligonucleotide strands or oligonucleotide. In one preferred embodiment, novel cross-linkages connect a nucleotide or nucleoside pentofuranosyl moiety on one strand with a nucleotide or nucleoside pentofuranosyl moiety on another strand. In another preferred embodiment, novel cross-linkages connect a nucleotide or nucleoside pentofuranosyl moiety on a first region of a single strand with a nucleotide or nucleoside pentofuranosyl moiety on a further region of the same strand. It is believed that such "sugar to sugar" cross-linkages were heretofore unknown. In a further embodiment of the invention, novel cross-linkages connect an abasic site on one oligonucleotide strand to a further site on another strand. Also, novel cross-linkages can connect an abasic site on a first region of a single strand with another site on another region of the single strand. It is believed that use of an abasic site as the attachment site for a oligonucleotide cross-linkage was also heretofore unknown.

In a further embodiment of the invention, novel cross-linkages of the invention connect a terminal sugar on one oligonucleotide strand or a first region of a single strand with a further site on a further strand or to a further region of a single strand.

One preferred cross-linkage of the invention connects the 2'-position of a ribose sugar on one oligonucleotide strand (or a first region of a single strand) to the 2'-position of a ribose sugar on another strand (or a further region of a single strand). This cross-linkage can be considered a 2'-ribose to 2'-ribose cross-linkage. A second preferred cross-linkage connects a 2'-ribose position on one oligonucleotide strand (or a first region of a single strand) to a C1'-ribose position (an abasic site) on another strand (or a further region of a single strand). This linkage can be considered a 2'-ribose to C1'-ribose cross-linkage. A third preferred cross-linkage connects a 2'-ribose position on one strand (or a first region of a single strand) to a seco-ribose on the 3'-terminal end of another strand (or a further region of a single strand). This linkage can be considered a 2'-ribose to seco-ribose linkage. A fourth preferred cross-linkage connects a 2'-ribose position on one strand (or a first region of a single strand) to 2' and 3' hydroxyls of a nucleoside sugar on the 3'-terminal end of another strand (or a further region of a single strand). This linkage can be considered a 2'-ribose to 2',3'-OH-ribose linkage.

For 2'-ribose to 2'-ribose cross-linkages, presently preferred are: cross-linkages between the 2'-hydroxyl groups (or other 2' substituent groups) of the nucleotide sugar moieties of a Watson/Crick nucleotide base pair; a cross-linkage between a 2'-hydroxyl group (or other 2' substituent group) of a nucleotide sugar moiety and a 2'-hydroxyl (or other 2' substituent group) of a nucleotide sugar moiety one base removed (in the 5'-direction) of the opposite strand; a cross-linkage between nucleotides one helical loop removed; or cross-linkage between nucleotides that are separated from each other along the strand or strands but that are located proximal or adjacent to each other in the higher-ordered structure. Molecular models and computer modeling indicate that the 2'-hydroxyls of RNA-RNA duplexes are exposed in the minor groove or minor side. Thus, direct covalent cross-linkage of the 2'-hydroxyls with a linker of appropriate length should not disturb the natural conformation of the duplexes.

As per the teachings of this invention, RNA-RNA, DNA-DNA or DNA-RNA type duplexes can be covalently cross-linked via a space-spanning group attached to nucleotide units at desired attachment sites. The length of the space-spanning group on each strand or region of a single strand can be between 1 to about 25 atoms. Thus, when two space-spanning groups are cross-linked together, the total length of the space-spanning groups, the cross-linker, and any other connecting atoms should be less than about 50 atoms in length.

The space-spanning groups are composed of carbon atoms or of carbon atoms plus one or more heteroatoms such as nitrogen, sulfur or oxygen. It is preferred that the space-spanning group on an individual strand or on a sequence region of a single strand be about 20 atoms or less in length. More preferably, the cross-linking group is from 8 to 13 atoms in length.

For 2'-ribose to C1'-ribose cross-linkages, a cross-linkage is formed between a 2'-position of a nucleotide sugar moiety on one strand (or on a first region of a single strand) and an abasic 1'-site on another strand (or on a further region of a single strand). The cross-linking group preferably is from 8 to 13 atoms in length.

For 2'-ribose to seco-ribose cross-linkages, a cross-linkage is formed between a 3'-terminal ribofuranosyl nucleoside of a first strand and a reactive functionality on a space-spanning group attached to the sugar moiety of a nucleotide on another strand (or on a further region of the first strand). Preferably, such cross-linkage is made through 2' and 3' hydroxyl groups of the 3'-terminal nucleoside. The 2' and 3' hydroxyl groups are first converted to a 2',3'-dialdehyde structure with periodate. A cross-linkage is formed upon hybridization with a complementary strand that has a connecting or space-spanning group bearing a functional group capable of reacting with the aldehydic functionality. If a 2'-aminoalkoxy connecting or space-spanning group is provided on the 2'-ribose strand, reaction of the aldehydic functionality of the seco-ribose strand should form a Schiff's base. Reduction of this Schiff's base with sodium cyanoborohydride affords a stable cross-linkage.

For 2'-ribose to 2',3'-OH-ribose cross-linkages, a cross-linkage is formed between 2' and 3' hydroxyls of a 3'-terminal ribofuranosyl nucleoside of a first strand and an aldehyde functionality on a space-spanning group attached to a nucleotide sugar moiety on another strand (or on a further region of the first strand). On hybridization of the strands (or strand), the aldehyde functionality on the space-spanning group reacts with the 2',3'-hydroxyls to form an acetal linkage.

Oligonucleotides in accordance with this invention preferably comprise from about 3 to about 100 subunits. It is preferred that such oligonucleotides comprise greater than about 6 subunits, more preferably from about 8 to about 60 subunits. Oligonucleotides having from about 10 to about 30 subunits are still more preferred. It will be recognized that a subunit comprises a base moiety linked to a sugar moiety. A subunit typically is bound to adjacent subunits through inter-nucleoside bonds. As indicated in the above-referenced United States patent applications, oligonucleotides have diagnostic, therapeutic, and prophylactic applications and also can be used as research reagents. For therapeutic use, cross-linked oligonucleotide analogs can be administered to animals, including humans, suffering from a disease state that it is desirous to treat.

It is generally preferred to apply a therapeutic agent of the invention internally such as orally, intravenously or intramuscularly with or without pharmacologically acceptable carriers. Other forms of administration, such as transdermal, topical or intralesional administration, may also be useful. Cross-linked oligonucleotides also can be included in suppositories.

In certain embodiments, a space-spanning group bearing an active functional group is located on one strand of a pair of duplexed oligonucleotide strands. Preferably, the space-spanning group is attached to a 2'-position of a nucleotide on the strand. The functional group is positioned on the space-spanning group at a point distal from the point at which the space-spanning group connects to the nucleotide. The space-spanning group is connected to the 2'-position via one or more connecting atoms. Preferably, the connecting atoms includes O, S, NH, CH₂ or CO.

The space-spanning group is preferably a linear or straight chain group. In one embodiment of the invention, the space-spanning group comprises a methylene chain from 1 to about 20 carbon atoms in length. Where the connecting atoms are methylene groups, the connecting group and the space-spanning group together form an alkyl chain. In other embodiments, one or more unsaturated sites are located in the space-spanning group, resulting in alkenyl or alkynyl chains. In these embodiments the alkenyl or alkynyl space-spanning groups have from 2 to about 20 carbon atoms.

In other embodiments the space-spanning group further includes branched chains and substituent groups that extend from a backbone or primary straight chain. Branched chains can be selected from branched alkyl, branched alkenyl or branched alkyne space-spanning groups. The branched chains, in addition to the carbon atoms of the main chain, have from 1 to about 30 carbon atoms. Further substituent atoms or substituent groups can also extend from the backbone or from any branched chains. Thus, the space-spanning group can include C₁ -C₂₀ straight chain alkyl, C₁ -C₂₀ straight chain substituted alkyl, C₂ -C₅₀ branched chain alkyl, C₂ -C₅₀ branched chain substituted alkyl, C₂ -C₂₀ straight chain alkenyl, C₂ -C₂₀ straight chain substituted alkenyl, C₃ -C₅₀ branched chain alkenyl, C₃ -C₅₀ branched chain substituted alkenyl, C₂ -C₂₀ straight chain alkyne, C₂ -C₂₀ straight chain substituted alkyne, C₃ -C₅₀ branched chain alkyne and C₃ -C₅₀ branched chain substituted alkyne groups. Other space-spanning groups include aryl, aralkyl and heterocyclic groups.

Representative examples of the space-spanning groups include but are not limited to methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl and eicosyl straight chained alkyl groups; 2-methylpropyl, 2-methyl-4-ethylbutyl, 2,4-diethylpropyl, 3-propylbutyl, 2,8-dibutyldecyl, 6,6-dimethyloctyl, 6-propyl-6-butyloctyl, 2-methylbutyl, 2-methylpentyl, 3-methylpentyl, 2-ethylhexyl, 3-penten-2-one, 3-methyl-2-butanol, 2-cyanooctyl, 3-methoxy-4-heptanal, 3-nitrobutyl, 4-isopropoxydodecyl, 4-azido-2-nitrodecyl, 5-mercaptononyl, 4-amino-1-pentenyl branched or substituted groups; allyl, crotyl, propargyl, 2-pentenyl unsaturated groups; 4-methylenebenzyl, 1,4-naphthyl, 2,7-anthracyl and 2,6-phenanthryl aryl or aralkyl groups.

Other space-spanning groups include polyamine, polyamide, polyester, polyethylene glycol and polyether, (polyalkoxyalkyl) groups. Polyamines include compounds of the structure amine functionality-(linker)_(m) !_(n). Normally the linker is a methylene chain. The amine functionalities can be primary amines, hydrazines, semicarbazides, thiosemicarbazides or similar nitrogenous species. The number of individual units of the polyamine (n) is selected in conjunction with the number of linker atoms (m) such that the total number of atoms is preferably 20 or less. Thus, if --HN(CH₂)_(m) --!_(n) is selected as the polyamine backbone, n could be from 1 to 10 depending on m. For example, if m is 1, n can range up to 10, and if m is 2, n can range up to 5. If larger nitrogenous species such as hydrazine or semicarbazide are utilized, n and m would be correspondingly smaller to reflect the increased number of atoms contributed by the nitrogenous species.

Polyamides, polyesters and polyethylene glycols according to the invention have structures analogous to the above-described polyamines, except that an amide, ester or alcohol functionality is substituted for the nitrogenous species of the polyamine. Polyether groups also have analogous structures, except that one or more ether oxygen atoms are interspersed in the carbon chains.

Aryl, aralkyl and heterocyclic space-spanning groups are similar to the above-described alkyl, alkenyl and alkynyl groups, except that appropriate aromatic, mixed alkyl-aromatic or heterocyclic groups are selected. Such space-spanning groups preferably are less than about 20 atoms in length, excluding atoms that are not directly in the space-spanning group chain but are within the structure of the aromatic or 5 heterocyclic ring. Aryl, aralkyl, and heterocyclic space-spanning groups can include up to about 50 atoms.

Substituent groups can be present on the above-described alkyl, alkenyl, alkyne, polyamine, polyamide, polyester, polyethylene glycol, polyether, aryl, aralkyl and heterocyclic space-spanning groups. Substituent groups include but are not limited to halogen, hydroxyl, keto, carboxy, nitrates, nitrites, nitro, nitroso, nitrile, trifluoromethyl, O-alkyl, S-alkyl, NH-alkyl, amino, azido, sulfoxide, sulfone, sulfide, silyl, intercalators, conjugates, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligonucleotides and groups that enhance the pharmacokinetic properties of oligonucleotides. Typical intercalators and conjugates include cholesterols, phospholipids, biotin, phenanthroline, phenazine, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Halogens include fluorine, chlorine, bromine and iodine. Groups that enhance pharmacodynamic properties, in the context of this invention, include groups that improve oligonucleotide uptake, enhance oligonucleotide resistance to degradation, and/or strengthen sequence-specific hybridization with RNA. Groups that enhance pharmacokinetic properties, in the context of this invention, include groups that improve oligonucleotide uptake, distribution, metabolism or excretion.

Nucleotides incorporating a connecting atom, a space-spanning group and a functional group on the end of the space-spanning group are generally synthesized as per the methods and syntheses disclosed in U.S. patent application Ser. Nos. 463,358, 566,977 and PCT/US91/00243. To introduce amine-functionalized space-spanning groups on a nucleotide within desired oligonucleotide sequences, 5'-dimethoxytrityl-2'-O, 2'-S or 2'-NH-(space-spanning group-N-phthalimido) nucleoside phosphoramidites are synthesized. The N-phthalimido amine protecting group is removed with concentrated NH₄ OH. N-phthalimido compounds thus provide an amine-functionalized space-spanning group attached to the 2'-position of nucleotide components of an oligonucleotide.

The following compounds for forming compounds having amine-functionalized space-spanning groups are commercially available from Aldrich Chemical Co., Inc., Milwaukee, Wis.: N-(2-bromoethyl)phthalimide, N-(3-bromopropyl)phthalimide and N-(4-bromobutyl)phthalimide. Other phthalimide-protected amine compounds can be conveniently synthesized from appropriate alkyl, aralkyl or aryl halides and phthalimide. Representative compounds include N-(7-bromoheptyl)phthalimide; N-(8-bromooctyl)phthalimide; N-(9-bromononyl)phthalimide; N-(10-bromododecyl)phthalimide; N-(10-bromoundecyl)phthalimide; N-(12-bromodocecyl)phthalimide; N-(13-bromotridecyl) phthalimide; N-(14-bromotetradecyl)phthalimide; N-(15-bromopentadecyl)phthalimide; N-(16-bromohexadecyl)-phthalimide; N-(17-bromoheptadecyl)phthalimide;N-(18-bromooctadecyl)phthalimide; N-(19-bromononadecyl)phthalimide; N-(3-bromo-2-methylpropyl)phthalimide; N-(4-bromo-2-methyl-3-ethylbutyl)phthalimide; N-(3-bromo-2,2-diethylpropyl)phthalimide; N-(4-bromo-3-propylbutyl)phthalimide; N-(10-bromo-2,8-dibutyldecyl)phthalimide; N-(8-bromo-6,6-dimethyloctyl)phthalimide;N-(8-bromo-6-propyl-6-butyloctyl)phthalimide; N-(4-bromo-2-methylbutyl)phthalimide; N-(5-bromo-2-methylpentyl)phthalimide; N-(5-bromo-3-methylpentyl)phthalimide; N-(6-bromo-2-ethylhexyl)phthalimide; N-(5-bromo-3-penten-2-one)phthalimide; N-(4-bromo-3-methyl-2-butanol)phthalimide; N-(8-bromo-3-amino-4-chloro-2-cyanooctyl)phthalimide; N-(7-bromo-3-methoxy-4-heptanal)phthalimide; N-(4-bromo-2-iodo-3-nitrobutyl)phthalimide; N-(12-bromo-4-isopropoxydodecyl)phthalimide; N-(10-bromo-4-azido-2-nitrodecyl)phthalimide;N-(9-bromo-5-mercaptononyl)phthal-imide; N-(5-bromo-4-aminopentenyl)phthalimide; N-(5-bromo-penten-2-yl)phthalimide; N-(3-bromoallyl)phthalimide; N-(4-bromocrotyl)phthalimide; N-(3-bromopropargyl)phthalimide; N-(1-bromonaphth-4-yl)phthalimide; N-(2-bromoanthrac-7-yl)phthalimide; and N-(2-bromophenanthr-6-yl)phthalimide. Such halide compounds are then reacted with an appropriate 2'-oxygen, 2'-sulfur or 2'-amine nucleoside.

In addition to amines (--NH₂), further preferred reactive moieties suitable for the functional groups of the invention include hydrazines (--NH--N--H--), hydroxylamines (--NH--OH), semicarbazides (--NH--C(O)--NH--NH₂), thiosemicarbazides (--NH--C(S)--NH--NH₂), hydrazones (--N═NH), hydrazides (--C(O)--NH--NH₂), alcohols (--OH), thiols (--SH), and aldehydes (--CH═O).

To introduce a nucleotide having a hydrazine-functionalized space-spanning group at its 2'-position into desired oligonucleotide sequences, 5'-dimethoxytrityl-2'-O--, -2'-S-- or -2'-NH-(space-spanning group-N-benzylcarbazide) nucleoside phosphoramidites are synthesized. Once an oligonucleotide incorporating these nucleotides is synthesized, the hydrazine functionality is generated by reduction of the carbazide with palladium on charcoal.

To introduce a nucleotide having a hydroxylamine-functionalized space-spanning group at its 2'-position within desired oligonucleotide sequences, 5'-dimethoxytrityl-2'-O--, -2'-S-- or 2'-NH-(space-spanning group-O-phthalimide)nucleoside phosphoramidites are synthesized. Once an oligonucleotide incorporating these nucleotides is synthesized, the hydroxylamine functionality is generated by treatment of the oligonucleotide with methylhydrazine. Semicarbazide and thiosemicarbazide functionalities on space-spanning groups can be introduced directly without protective groups. 5'-Dimethoxytrityl-2'-O--, 2'-S-- or 2'-NH-(alkylsemicarbazide)-nucleoside phosphoramidites are synthesized and incorporated into desired oligonucleotides to provide a semicarbazide functionalized space-spanning group attached to the 2'-position of a component nucleotide of an oligonucleotide.

Hydrazide functionalities are formed on space-spanning groups via reaction of hydrazine with an nucleotide bearing a 2'-O-alkylester. Alcohol functionalities can be formed on space-spanning groups via Ω-bromo alcohols. The alcohol hydroxyl group is protected with a tetrahydropyran (THP) blocking group. The THP- protected compound is then reacted with an activated nucleoside to attach the THP-protected compound at the 2'-position of the nucleoside. After oligonucleotide synthesis, the THP blocking group is removed to expose the alcoholic functionality. Thiol functionalities can be directly formed via ring opening of a 2,2'-anhydro pyrimidine nucleoside using α,Ω-dithiol compounds. Alternatively, the thiol functionality of space-spanning compounds can be protected with S-trityl protecting groups. After oligonucleotide synthesis, the S-trityl protecting groups are removed with silver nitrate.

Aldehydes used as active functional groups in the formation of cross-linkages can be "masked" as protected aldehydes or aldehyde precursors during initial oligonucleotide synthesis. The aldehyde is generated prior to cross-linking from the protected aldehyde or aldehyde precursor and is utilized as one of the reactants for the formation of a covalent cross-linkage. Representative protected aldehydes are C-formyl, o-methylaminobenzenethio, aryl substituted imidazolidino moieties such as di-phenylimidazolidino, dithianes, bis(p-nitrophenoxy) and acetals. Particularly preferred acetals are di-aralkyl acetals such as bis(o-nitrobenzyl) acetals that are removed via catalytic hydrogenolysis. The diphenylimidazolidino group, which is removed via hydrazine hydrate/sodium cyanoborohydride treatment, is also particularly preferred. Aldehyde precursors include nitriles, alcohols and esters. Nitriles and esters are reduced to their corresponding aldehydes, while alcohols are oxidized to their corresponding aldehydes. Nitriles can be treated with hydrazine, followed by reduction with sodium cyanoborohydride. Esters can be reduced with lithium aluminum hydride. Alcohols can be oxidized under mild oxidation conditions such as Pfitzner-Moffat conditions. See, J. Am. Chem. Soc., 1965, 87, 5661. Such conditions have been used by Hansske, et al., Tetrahedron, 1984, 40, 125, to oxidize 2' or 3' hydroxyl groups of various nucleosides using both a chromic acid oxidation reagent as well as a DMSO/Ac₂ O oxidation reagent.

Aldehyde precursors and protected aldehydes can be used both at the nucleoside/nucleotide level and at the oligonucleotide level. Individual nucleotides (or their nucleoside precursors) incorporating an aldehyde precursor or protected aldehyde can be directly incorporated into an oligonucleotide. Alternately, an aldehyde precursor can be converted to a protected aldehyde, as with C-formyl protected aldehydes, and then directly incorporated into an oligonucleotide. Upon synthesis of the oligonucleotide, the aldehyde is regenerated from the protected aldehyde or generated from the aldehyde precursor and the oligonucleotide strand is cross-linked with a further oligonucleotide strand or with a further region of the same oligonucleotide strand. Use of aldehyde precursors is suggested for certain oligonucleotides, such as mimics of pyrimidine tract sequences located upstream from gene splice junctions. One such pyrimidine tract sequence includes the sequence CCC CTC CCC. Since such tracts only contain pyrimidine nucleotides, acidic oxidation conditions used for conversion of alcohols to aldehydes will not result in acidic de-purination or acidic strand cleavage of the oligonucleotide.

As will be recognized, hydrazine, hydroxylamine, semicarbazide, thiosemicarbazide, hydrazone, hydrazide, alcohol or thiol functional groups on one space-spanning group can be reacted with an aldehydic group (a --CH═O group) on a further space-spanning group as, for example, in the above-described embodiment wherein a 2'-ribose to 2'-ribose linkage is formed. These functional groups also can be reacted with an aldehydic group at the C1'-position of an abasic site to form a 2'-ribose to C1'-ribose linkage. Alternatively, these functional groups can be reacted with one of the aldehydic groups of a 2',3'-dialdehyde 3'-terminal nucleoside to form a 2'-ribose to a seco-ribose linkage, or to form imine (--C═N--), hydrazone (--C═N--NH--), oxime (--O--N═CH--), semicarbazone (NH--C(O)--NH--N═C--), thiosemicarbazone (NH--C(S)--NH--N═C--), azine (--H═N--N═CH--), hydrazide-hydrazone (--C(O)--NH--N═C--), hemiacetal (--CH(OH)--O--CH₂ --) or thiohemiacetal (--CH(OH)--S--CH₂ --) cross-linkages.

When an aldehyde on a first strand reacts with an alcohol or a thioacetal on another strand or on a further region of the first strand, the resulting hemiacetal or thiohemiacetal theoretically can react with a further alcohol or thiol group. However, such reaction generally is not kinetically (entropically) favorable, as it usually occurs in a water solution or under other conditions in which the concentration of the further alcohol or thiol group is low.

In those embodiments of the invention wherein a cross-linkage is formed between a 2'-ribose on a first strand (or a first region of a single strand) and a 2'-ribose on another strand (or a further region of the single strand), space-spanning groups and reactive functionalities are attached to each of the strands (or regions of a single strand). The space-spanning groups and reactive functionalities are attached to the 2'-position of each of the strands (or regions of a single strand), as above, utilizing connecting atoms. The strand or strands are then cross-linked by reaction of the reactive functionalities. Accordingly, the reactive functionalities can each be considered "bond precursors" or "bond means" since they together form a covalent bond.

Depending upon the reactive functionality on each of the strands (or regions of a single strand), a covalent bond will be formed as an integral part of the cross-linkage. When a thiol group is the active functionality on each of the strands, oxidization results in a disulfide cross-linkage. When an aldehyde is the functional group on one strand (or a first region on a single strand) and an amine is the functional group on the further strand (or further region on a single strand), an imine (--N═CH--, Schiff's base) cross-linkage results. The imine cross-linkage can be reduced to an amine (--NH--CH₂ --) using a reducing agent such as sodium cyanoborohydride.

Reaction of a hydrazine (--NH--NH--) with an aldehyde yields a hydrazone (--H═N--NH--) cross-linkage, while reaction of a hydroxylamine (HO--N--) with an aldehyde yields an oxime (--O--N═CH--) cross-linkage. Reaction of an aldehyde with a hydrazide (--C(O)--NH--NH₂) yields a hydrazide-hydrazone (--C(O)--NH--N═CH--) cross-linkage, reaction of an aldehyde with a semicarbazide (--NH--C(O)--NH--NH₂) yields a semicarbazone (--NH--C(O)--NH--N═C--) cross-linkage, and reaction of an aldehyde with a thiosemicarbazide (--NH--C(S)--NH--NH₂) yields a thiosemicarbazone (--NH--C(S)--NH--N═C--) cross-linkage. The hydrazone, oxime, hydrazide-hydrazone, semicarbazone and thiosemicarbazone linkages can be reduced to corresponding hydrazine, hydroxylamine, hydrazide-hydrazine, semicarbazide and thiosemicarbazide, respectively, if desired.

The active functional groups on the space-spanning moieties can be reacted with both heterobifunctional and homobifunctional groups. As will be recognized, formaldehyde is a homobifunctional group when it is reacted with two amines. Reaction of formaldehyde with amine functional groups that are attached to each of two strands (or regions of a single strand) yields an aminoalkylamine (--NH--CH₂ --NH--) linkage.

Hydrazine also can be used as a bifunctional group to join strands that each have a space-spanning group terminated with an aldehyde. The hydrazine adds to a first aldehyde group to form an aldehyde-hydrazone that, in turn, reacts with the second aldehyde to yield an azine (--CH═N--N═CH--) cross-linkage.

Heterobifunctional groups allow the use of one active functionality on one strand (or a region of a single strand) and the use of a different active functionality on another strand (or another region of a single strand). Thus, one strand could have an amine functionality on an end of its space-spanning group and another strand could have a thiol functionality on the end of its space-spanning group.

Space-spanning groups on one of the strands (or region of a single strand) may not be necessary where heterobifunctional groups or certain of the above-noted active functionalities are employed. An attaching group, for example a 2'-amino or 2'-thio of a 2'-substituted-2'-deoxy-erythro-pentofuranosyl moiety, could be used directly as an active functionality. The space-spanning group and its active functionality from the other strand could react directly with this 2'-positioned active functionality. For example a 2'-deoxy-2'-thio-erythro-pentofuranosyl sugar could be utilized as the sugar moiety of a selected nucleotide on one strand. An omega thiol group on the space-spanning group of a complementary duplex strand can then be coupled to the first 2'-thio group via a disulfide linkage. Other 2' groups suitable for this type of cross-linkage include 2'-amino groups. A 2'-keto group also could be reacted with a suitable hydrazine or other reactive functionality.

A variety of heterobifunctional coupling reagents are available from commercial sources including Pierce (Rockford, Ill.). These reagents include 1-ethyl-3-(3-dimethylaminopropyl)carbodimide (a water soluble DCC type coupling reagent), bifunctional imidoesters such as dimethyl adipimidate dihydrochloride, dimethyl pimelimidate dihydrochloride, and dimethyl 3,3'-dithiobispropionimidate dihydrochloride (for coupling amine groups), and 2-iminothiolane (for coupling amines thiols). Coupling reagents also include compounds having N-hydroxysuccinimide moieties that react with amines, compounds having internal disulfide bonds that can be released to expose an active thiol moiety that can be further coupled via a disulfide bond with a further thiol functionality, and compounds having sulfosuccinimidyl groups (e.g., N-hydroxysulfosuccinimide) that have different reactivity toward amines than N-hydroxysuccinimides. Coupling reagents further include pyridyl-disulfides that exchange with free thiol moieties, active halogen species such as β-carbonyl alkylhalides, e.g. iodo acetamide, maleimide moieties and photoreactive aryl azides such as phenyl azide. Also, thioisocyanates can be used to couple with active functionalities on space-spanning groups.

The various functional groups of the bifunctional cross-linkers are joined together with suitable linking atoms. Representative, commercially available bifunctional compounds include sulfosuccinimidyl 4-(N-maleimidoethyl)cyclohexane-1-carboxylate, N-succinimidyl 3-(2-pyridyldithio)propionate, N-sulfosuccinimidyl(4-iodoacetyl)aminobenzoate, sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate, m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester, succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate,bismaleimidohexane, disulfosuccinimidyl tartrate, ethylene glycolbis(succinimidylsuccinate), dithiobis(succinimidylpropionate), disuccinimidyl suberate, N-hydroxysuccinimidyl-4-azidosalicylic acid, N-5-azido-2-nitrobenzoyloxysuccinimide and sulfonsuccinimidyl 2-(m-azido-o-nitrobenzamido)-ethyl-1,3'-dithiopropionate. Each of these bifunctional groups are reacted with an appropriate active functionality on the space-spanning group. Thus, oligonucleotide strands (or regions of a single strand) bearing the space-spanning groups are cross-linked through the bifunctional group.

The 6-bromo-5,5-dimethoxyhexanohydrazide and N-hydroxysuccinimidyl iodoacetate reagents disclosed by Summerton, et al. and Meyer, et al., respectively, in the references cited above can also be used as heterobifunctional cross-linking reagents. The hydrazine moiety of the Summerton reagent and the succinimidyl moiety of the Meyer reagent can be linked to one strand of duplex strands and the other strand can be linked to the bromoketone or the iodoacetamido portions of these reagents. For instance, the hydrazine functionality of the Summerton reagent would first be reacted with an aldehydic functionality on one strand, the α-haloketal functionality of the reagent would be activated to an α-haloketone via pH adjustment, and the α-haloketone would then be further reacted with an amine or thiol functionality on the other strand. The active succinimide functionality of the Meyer reagent would be linked to a first amine functionality on a first strand and the resulting iodoacetamidoalkyl functionality would be linked to a further amine or thiol functionality on the other strand.

Since both the Summerton and Meyer reagents can be used to join two amine functionalities (two hydrazine functionalities in the case of the Summerton reagent), they can also be considered homobifunctional linking reagents. Neither the Summerton reagent nor the Meyer reagent are used in accordance with the invention to couple duplex strands via the 7-position of a purine ring. Thus, no charged (i.e., chemically unstable) species are formed but, rather uncharged, chemically stable cross-linkages of the structures --CH₂ --S--CH₂ --C(O)--CH₂ -- and --CH₂ --NH--CH₂ --C(O)--CH₂ --.

By terminating a first oligonucleotide strand at its 3' end with a terminal ribofuranosyl nucleoside, cross-linking to a further oligonucleotide strands (or to a further region of a single strand) can be effected via one of two mechanisms. The first mechanism involves generation of a seco ribose by, for example, periodate oxidation of the 2',3' hydroxyl groups of the 3'-terminal ribose. See, e.g., Lemaitre, Proc. Natl. Acad. Sci. USA, 1986, 84, 648; Chang, Biochem. J., 1981, 299, 281; and Easterbrook-Smith, Eur. J. Biochem, 1976, 62, 125.! Such oxidation yields a 2',3' di-aldehyde (a seco ribose), which is reacted with a second strand (or a further region of a single strand) that includes a space-spanning group bearing an active amine functionality. Such reaction yields a Schiff's base, which is reduced with, for example, sodium cyanoborohydride, to form an amine cross-linker having an amine group directly coupled to the seco ribose of the 3'-terminal nucleoside of the first oligonucleotide strand.

The second mechanism involves formation of an acetal cross-linkage from the 2',3'-hydroxyl groups of the terminal ribose. To form the acetal linkage, the hydroxyl groups of the terminal ribose are reacted with an aldehyde group on a space-spanning group on the second strand (or further region of a single strand). Acetals of this type have been utilized to attach a nitrogen mustard to a terminal ribose, as described by Knorre, et al., Nucleotide and Oligonucleotide Derivatives as Enzyme-and Nucleic Acid Targeted irreversible Inhibitors, Chemical Aspects, G. Weber (Ed.), Advances Press, Oxford, 1986, pp. 277-299. The nitrogen mustard was then further reacted with a complementary sequence of a short oligonucleotide. Significantly, the cross-linkage was formed via the nitrogen mustard, not via the acetal. As noted above, cross-linkages formed via nitrogen mustards generate charged species that are chemically unstable. The present invention employs an acetal, which does not generate a charged species, to effect cross-linking.

Taken together, the space-spanning groups, the functional groups, and the connecting atoms comprise a moiety selected from the group consisting of: C₁ -C₂₀ straight chain alkylamine, C₁₁ -C₂₀ straight chain O-alkylamine, C₁ -C₂₀ straight chain S-alkylamine, C₁ -C₂₀ straight chain NH-alkylamine, C₁ -C₂₀ straight chain substituted alkylamine, C₁ -C₂₀ straight chain substituted O-alkylamine, C₁ -C₂₀ straight chain substituted S-alkylamine, C₁ -C₂₀ straight chain substituted NH-alkylamine, C₂ -C₅₀ branched chain alkylamine, C₂ -C₅₀ branched chain O-alkylamine, C₂ C₅₀ branched chain S-alkylamine, C₂ -C₅₀ branched chain NH-alkylamine, C₂ -C₅₀ branched chain substituted alkylamine, C₂ -C₅₀ branched chain substituted O-alkylamine, C₂ -C₅₀ branched chain substituted S-alkylamine, C₂ -C₅₀ branched chain substituted NH-alkylamine, C₂ -C₂₀ straight chain alkenylamine, C₂ -C₂₀ straight chain O-alkenylamine, C₂ -C₂₀ straight chain S-alkenylamine, C₂ -C₂₀ straight chain NH-alkenylamine, C₂ -C₂₀ straight chain substituted alkenylamine, C₂ -C₂₀ straight chain substituted O-alkenylamine, C₂ -C₂₀ straight chain substituted S-alkenylamine, C₂ -C₂₀ straight chain substituted NH-alkenylamine, C₃ -C₅₀ branched chain alkenylamine, C₃ -C₅₀ branched chain O-alkenylamine, C₃ -C₅₀ branched chain S-alkenylamine, C₃ -C₅₀ branched chain NH-alkenylamine, C₃ -C₅₀ branched chain substituted alkenylamine, C₃ -C₅₀ branched chain substituted O-alkenylamine, C₃ -C₅₀ branched chain substituted S-alkenylamine, C₃ -C₅₀ branched chain substituted NH-alkenylamine, C₂ -C₂₀ straight chain alkynylamine, C₂ -C₂₀ straight chain O-alkynylamine, C₂ -C₂₀ straight chain S-alkynylamine, C₂ -C₂₀ straight chain NH-alkynylamine, C₂ -C₂₀ straight chain substituted alkynylamine, C₂ -C₂₀ straight chain substituted O-alkynylamine, C₂ -C₂₀ straight chain substituted S-alkynylamine, C₂ -C₂₀ straight chain substituted NH-alkynylamine, C₃ -C₅₀ branched chain alkynylamine, C₂ -C₅₀ branched chain O-alkynylamine, C₃ -C₅₀ branched chain S-alkynylamine, C₃ -C₅₀ branched chain NH-alkynylamine, C₃ -C₅₀ branched chain substituted alkynylamine, C₃ -C₅₀ branched chain substituted O-alkynylamine, C₃ -C₅₀ branched chain substituted S-alkynylamine, C₃ -C₅₀ branched chain substituted NH-alkynylamine, C₁ -C₂₀ straight chain alkylhydrazine, C₁ -C₂₀ straight chain O-alkylhydrazine, C₁ -C₂₀ straight chain S-alkylhydrazine, C₁ -C₂₀ straight chain NH-alkylhydrazine, C₁ -C₂₀ straight chain substituted alkylhydrazine, C₁ -C₂₀ straight chain substituted O-alkylhydrazine, C₁ -C₂₀ straight chain substituted S-alkylhydrazine, C₁ -C₂₀ straight chain substituted NH-alkylhydrazine, C₂ -C₅₀ branched chain alkylhydrazine, C₂ -C₅₀ branched chain O-alkylhydrazine, C₂ -C₅₀ branched chain S-alkylhydrazine, C₂ -C₅₀ branched chain NH-alkylhydrazine, C₂ -C₅₀ branched chain substituted alkylhydrazine, C₂ -C₅₀ branched chain substituted O-alkylhydrazine, C₂ -C₅₀ branched chain substituted S-alkylhydrazine, C₂ -C₅₀ branched chain substituted NH-alkylhydrazine, C₂ -C₂₀ straight chain alkenylhydrazine, C₂ -C₂₀ straight chain O-alkenylhydrazine, C₂ -C₂₀ straight chain S-alkenylhydrazine, C₂ -C₂₀ straight chain NH-alkenylhydrazine, C₂ -C₂₀ straight chain substituted alkenylhydrazine, C₂ -C₂₀ straight chain substituted O-alkenylhydrazine, C₂ -C₂₀ straight chain substituted S-alkenylhydrazine, C₂ -C₂₀ straight chain substituted NH-alkenylhydrazine, C₃ -C₅₀ branched chain alkenylhydrazine, C₃ -C₅₀ branched chain O-alkenylhydrazine, C₃ -C₅₀ branched chain S-alkenylhydrazine, C₃ -C₅₀ branched chain NH-alkenylhydrazine, C₃ -C₅₀ branched chain substituted alkenylhydrazine, C₃ -C₅₀ branched chain substituted O-alkenylhydrazine, C₃ -C₅₀ branched chain substituted S-alkenylhydrazine, C₃ -C₅₀ branched chain substituted NH-alkenylhydrazine, C₂ -C₂₀ straight chain alkynylhydrazine, C₂ -C₂₀ straight chain O-alkynylhydrazine, C₂ -C₂₀ straight chain S-alkynylhydrazine, C₂ -C₂₀ straight chain NH-alkynylhydrazine, C₂ -C₂₀ straight chain substituted alkynylhydrazine, C₂ -C20 straight chain substituted O-alkynylhydrazine, C₂ -C₂₀ straight chain substituted S-alkynylhydrazine, C₂ -C₂₀ straight chain substituted NH-alkynylhydrazine, C₃ -C₅₀ branched chain alkynylhydrazine, C₂ -C₅₀ branched chain O-alkynylhydrazine, C₃ -C₅₀ branched chain S-alkynylhydrazine, C₃ -C₅₀ branched chain NH-alkynylhydrazine, C₃ -C₅₀ branched chain substituted alkynylhydrazine, C₃ -C₅₀ branched chain substituted O-alkynylhydrazine, C₃ -C₅₀ branched chain substituted S-alkynylhydrazine, C₃ -C₅₀ branched chain substituted NH-alkynylhydrazine, C₁ -C₂₀ straight chain alkylhydroxylamine, C₁ -C₂₀ straight chain O-alkylhydroxylamine, C₁ -C₂₀ straight chain S-alkylhydroxylamine, C₁ -C₂₀ straight chain NH-alkylhydroxylamine, C₁ -C₂₀ straight chain substituted alkylhydroxylamine, C₁ -C₂₀ straight chain substituted O-alkylhydroxylamine, C₁ -C₂₀ straight chain substituted S-alkylhydroxylamine, C₁ -C₂₀ straight chain substituted NH-alkylhydroxylamine, C₂ -C₅₀ branched chain alkylhydroxylamine, C₂ -C₅₀ branched chain O-alkylhydroxylamine, C₂ -C₅₀ branched chain S-alkylhydroxylamine, C₂ -C₅₀ branched chain NH-alkylhydroxylamine, C₂ -C₅₀ branched chain substituted alkylhydroxylamine, C₂ -C₅₀ branched chain substituted O-alkylhydroxylamine, C₂ -C₅₀ branched chain substituted S-alkylhydroxylamine, C₂ -C₅₀ branched chain substituted NH-alkylhydroxylamine, C₂ -C₂₀ straight chain alkenylhydroxylamine, C₂ -C₂₀ straight chain O-alkenylhydroxylamine, C₂ -C₂₀ straight chain S-alkenylhydroxylamine, C₂ -C₂₀ straight chain NH-alkenylhydroxylamine, C₂ -C₂₀ straight chain substituted alkenylhydroxylamine, C₂ -C₂₀ straight chain substituted O-alkenylhydroxylamine, C₂ -C₂₀ straight chain substituted S-alkenylhydroxylamine, C₂ -C₂₀ straight chain substituted NH-alkenylhydroxylamine, C₃ -C₅₀ branched chain alkenylhydroxylamine, C₃ -C₅₀ branched chain O-alkenylhydroxylamine, C₃ -C₅₀ branched chain S-alkenylhydroxylamine, C₃ -C₅₀ branched chain NH-alkenylhydroxylamine, C₃ -C₅₀ branched chain substituted alkenylhydroxylamine, C₃ -C₅₀ branched chain substituted O-alkenylhydroxylamine, C₃ -C₅₀ branched chain substituted S-alkenylhydroxylamine, C₃ -C₅₀ branched chain substituted NH-alkenylhydroxylamine, C₂ -C₂₀ straight chain alkynylhydroxylamine, C₂ -C₂₀ straight chain O-alkynylhydroxylamine, C₂ -C₂₀ straight chain S-alkynylhydroxylamine, C₂ -C₂₀ straight chain NH-alkynylhydroxylamine, C₂ -C₂₀ straight chain substituted alkynylhydroxylamine, C₂ -C₂₀ straight chain substituted O-alkynylhydroxylamine, C₂ -C₂₀ straight chain substituted S-alkynylhydroxylamine, C₂ -C₂₀ straight chain substituted NH-alkynylhydroxylamine, C₃ -C₅₀ branched chain alkynylhydroxylamine, C₂ -C₅₀ branched chain O-alkynylhydroxylamine, C₃ -C₅₀ branched chain S-alkynylhydroxylamine, C₃ -C₅₀ branched chain NH-alkynylhydroxylamine, C₃ -C₅₀ branched chain substituted alkynylhydroxylamine, C₃ -C₅₀ branched chain substituted O-alkynylhydroxylamine, C₃ -C₅₀ branched chain substituted S-alkynylhydroxylamine, C₃ -C₅₀ branched chain substituted NH-alkynylhydroxylamine, C₁ -C₂₀ straight chain alkylsemicarbazide, C₁ -C₂₀ straight chain O-alkylsemicarbazide, C₁ -C₂₀ straight chain S-alkylsemicarbazide, C₁ -C₂₀ straight chain NH-alkylsemicarbazide, C₁ -C₂₀ straight chain substituted alkylsemicarbazide, C₁ -C₂₀ straight chain substituted O-alkylsemicarbazide, C₁ -C₂₀ straight chain substituted S-alkylsemicarbazide, C₁ -C₂₀ straight chain substituted NH-alkylsemicarbazide, C₂ -C₅₀ branched chain alkylsemicarbazide, C₂ -C₅₀ branched chain O-alkylsemicarbazide, C₂ -C₅₀ branched chain S-alkylsemicarbazide, C₂ -C₅₀ branched chain NH-alkylsemicarbazide, C₂ -C₅₀ branched chain substituted alkylsemicarbazide, C₂ -C₅₀ branched chain substituted O-alkylsemicarbazide, C₂ -C₅₀ branched chain substituted S-alkylsemicarbazide, C₂ -C₅₀ branched chain substituted NH-alkylsemicarbazide, C₂ -C₂₀ straight chain alkenylsemicarbazide, C₂ -C₂₀ straight chain O-alkenylsemicarbazide, C₂ -C₂₀ straight chain S-alkenylsemicarbazide, C₂ -C₂₀ straight chain NH-alkenylsemicarbazide, C₂ -C₂₀ straight chain substituted alkenylsemicarbazide, C₂ -C₂₀ straight chain substituted O-alkenylsemicarbazide, C₂ -C_(straight) chain substituted S-alkenylsemicarbazide, C₂ -C₂₀ straight chain substituted NH-alkenylsemicarbazide, C₃ -C₅₀ branched chain alkenylsemicarbazide, C₃ -C₅₀ branched chain O-alkenylsemicarbazide, C₃ -C₅₀ branched chain S-alkenyl-semicarbazide, C₃ -c₅₀ branched chain NH-alkenylsemicarbazide, C₃ -C₅₀ branched chain substituted alkenylsemicarbazide, C₃ -C₅₀ branched chain substituted O-alkenylsemicarbazide, C₃ -C₅₀ branched chain substituted S-alkenylsemicarbazide, C₃ -C₅₀ branched chain substituted NH-alkenylsemicarbazide, C₂ -C₂₀ straight chain alkynylsemicarbazide, C₂ -C₂₀ straight chain O-alkynylsemicarbazide, C₂ -C₂₀ straight chain S-alkynylsemicarbazide, C₂ -C₂₀ straight chain NH-alkynylsemicarbazide, C₂ -C₂₀ straight chain substituted alkynylsemicarbazide, C₂ -C₂₀ straight chain substituted O-alkynylsemicarbazide, C₂ -C₂₀ straight chain substituted S-alkynylsemicarbazide, C₂ -C₂₀ straight chain substituted NH-alkynylsemicarbazide, C₃ -C₅₀ branched chain alkynylsemicarbazide, C₂ -C₅₀ branched chain O-alkynylsemicarbazide, C₃ -C₅₀ branched chain S-alkynylsemicarbazide, C₃ -C₅₀ branched chain NH-alkynylsemicarbazide, C₃ -C₅₀ branched chain substituted C₃ -C₅₀ branched chain substituted O-alkynylsemicarbazide C₃ -C₅₀ branched chain substituted S-alkynylsemicarbazide, C₃ -C₅₀ branched chain substituted NH-alkynylsemicarbazide, C₁ -C₂₀ straight chain alkylthiosemicarbazide, C₁ -C₂₀ straight chain O-alkylthiosemicarbazide, C₁ -C₂₀ straight chain S-alkylthiosemicarbazide, C₁ -C₂₀ straight chain NH-alkylthiosemicarbazide, C_(-C) ₂₀ straight chain substituted alkylthiosemicarbazide, C₁ -C₂₀ straight chain substituted O-alkylthiosemicarbazide, C₁ -C₂₀ straight chain substituted S-alkylthiosemicarbazide, C₁ -C₂₀ straight chain substituted NH-alkylthiosemicarbazide, C₂ -C₅₀ branched chain alkylthiosemicarbazide, C₂ -C₅₀ branched chain O-alkylthiosemicarbazide, C₂ -C₅₀ branched chain S-alkylthiosemicarbazide, C₂ -C₅₀ branched chain NH-alkylthiosemicarbazide, C₂ -C₅₀ branched chain substituted alkylthiosemicarbazide, C₂ -C₅₀ branched chain substituted O-alkylthiosemicarbazide, C₂ -C₅₀ branched chain substituted S-alkylthiosemicarbazide, C₂ -C₅₀ branched chain substituted NH-alkylthiosemicarbazide, C₂ -C₂₀ straight chain alkenylthiosemicarbazide, C₂ -C₂₀ straight chain O-alkenylthiosemicarbazide, C₂ -C₂₀ straight chain S-alkenylthiosemicarbazide, C₂ -C₂₀ straight chain NH-alkenylthiosemicarbazide, C₂ -C₂₀ straight chain substituted alkenylthiosemicarbazide, C₂ -C₂₀ straight chain substituted O-alkenylthiosemicarbazide, C₂ -C₂₀ straight chain substituted S-alkenylthiosemicarbazide, C₂ -C₂₀ straight chain substituted NH-alkenylthiosemicarbazide, C₃ -C₅₀ branched chain alkenylthiosemicarbazide, C₃ -C₅₀ branched chain O-alkenylthiosemicarbazide, C₃ -C₅₀ branched chain S-alkenylthiosemicarbazide, C₃ -C₅₀ branched chain NH-alkenylthiosemicarbazide, C₃ -C₅₀ branched chain substituted alkenylthiosemicarbazide, C₃ -C₅₀ branched chain substituted O-alkenylthiosemicarbazide, C₃ -C₅₀ branched chain substituted S-alkenylthiosemicarbazide, C₃ -C₅₀ branched chain substituted NH-alkenylthiosemicarbazide, C₂ -C₂₀ straight chain alkynylthiosemicarbazide, C₂ -C₂₀ straight chain O-alkynylthiosemicarbazide, C₂ -C₂₀ straight chain S-alkynylthiosemicarbazide, C₂ -C₂₀ straight chain NH-alkynylthiosemi-carbazide, C₂ -C₂₀ straight chain substituted alkynylthiosemicarbazide, C₂ -C₂₀ straight chain substituted O-alkynylthiosemicarbazide, C₂ -C₂₀ straight chain substituted S-alkynylthiosemicarbazide, C₂ -C₂₀ straight chain substituted NH-alkynylthiosemicarbazide, C₃ -C₅₀ branched chain alkynylthiosemicarbazide, C₂ -C₅₀ branched chain O-alkynylthiosemicarbazide, C₃ -C₅₀ branched chain S-alkynylthiosemicarbazide, C₃ -C₅₀ branched chain NH-alkynylthiosemicarbazide, C₃ -C₅₀ branched chain substituted alkynylthiosemicarbazide, C₃ -C₅₀ branched chain substituted O-alkynylthiosemicarbazide, C₃ -C₅₀ branched chain substituted S-alkynylthiosemicarbazide, C₃ -C₅₀ branched chain substituted NH-alkynylthiosemicarbazide, C₁ -C₂₀ straight chain alkylhydrazone, C₁ -C₂₀ straight chain O-alkylhydrazone, C₁ -C₂₀ straight chain S-alkylhydrazone, C₁ -C₂₀ straight chain NH-alkylhydrazone, C₁ -C₂₀ straight chain substituted alkylhydrazone, C₁ -C₂₀ straight chain substituted O-alkylhydrazone, C₁ -C₂₀ straight chain substituted S-alkylhydrazone, C₁ -C₂₀ straight chain substituted NH-alkylhydrazone, C₂ -C₂₀ branched chain alkylhydrazone, C₂ -C₅₀ branched chain O-alkylhydrazone, C₂ -C₅₀ branched chain S-alkylhydrazone, C₂ -C₅₀ branched chain NH-alkylhydrazone, C_(-C) ₅₀ branched chain substituted alkylhydrazone, C₂ -C₅₀ branched chain substituted O-alkylhydrazone, C₂ -C₅₀ branched chain substituted S-alkylhydrazone, C₂ -C₅₀ branched chain substituted NH-alkylhydrazone, C₂ -C₂₀ straight chain alkenylhydrazone, C₂ -C₂₀ straight chain O-alkenylhydrazone, C₂ -C₂₀ straight chain S-alkenylhydrazone, C₂ -C₂₀ straight chain NH-alkenylhydrazone, C₂ -C₂₀ straight chain substituted alkenylhydrazone, C₂ -C₂₀ straight chain substituted O-alkenylhydrazone, C₂ -C₂₀ straight chain substituted S-alkenylhydrazone, C₂ -C₂₀ straight chain substituted NH-alkenylhydrazone, C₃ -C₅₀ branched chain alkenylhydrazone, C₃ -C₅₀ branched chain O-alkenylhydrazone, C₃ -C₅₀ branched chain S-alkenylhydrazone, C₃ -C₅₀ branched chain NH-alkenylhydrazone, C₃ -C₅₀ branched chain substituted alkenylhydrazone, C₃ -C₅₀ branched chain substituted O-alkenylhydrazone, C₃ -C₅₀ branched chain substituted S-alkenylhydrazone, C₃ -C₅₀ branched chain substituted NH-alkenylhydrazone, C₂ -C₂₀ straight chain alkynylhydrazone, C₂ -C₂₀ straight chain O-alkynylhydrazone, C₂ -C₂₀ straight chain S-alkynylhydrazone, C₂ -C₂₀ straight chain NH-alkynylhydrazone, C₂ -C₂₀ straight chain substituted alkynylhydrazone, C₂ -C₂₀ straight chain substituted O-alkynylhydrazone, C₂ -C₂₀ straight chain substituted S-alkynylhydrazone, C₂ -C₂₀ straight chain substituted NH-alkynylhydrazone, C₃ -C₅₀ branched chain alkynylhydrazone, C₂ -C₅₀ branched chain O-alkynylhydrazone, C₃ -C₅₀ branched chain S-alkynylhydrazone, C₃ -C₅₀ branched chain NH-alkynylhydrazone, C₃ -C₅₀ branched chain substituted alkynylhydrazone, C₃ -C₅₀ branched chain substituted O-alkynylhydrazone, C₃ -C₅₀ branched chain substituted S-alkynylhydrazone, C₃ -C₅₀ branched chain substituted NH-alkynylhydrazone, C₁ -C₂₀ straight chain alkylhydrazide, C₁ -C₂₀ straight chain O-alkylhydrazide, C₁ -C₂₀ straight chain S-alkylhydrazide, C₁ -C₂₀ straight chain NH-alkylhydrazide, C₁ -C₂₀ straight chain substituted alkylhydrazide, C₁ -C₂₀ straight chain substituted O-alkylhydrazide, C₁ -C₂₀ straight chain substituted S-alkylhydrazide, C₁ -C₂₀ straight chain substituted NH-alkylhydrazide, C₂ -C₅₀ branched chain alkylhydrazide, C₂ -C₅₀ branched chain O-alkylhydrazide, C₂ -C₅₀ branched chain S-alkylhydrazide, C₂ -C₅₀ branched chain NH-alkylhydrazide, C₂ -C₅₀ branched chain substituted alkylhydrazide, C₂ -C₅₀ branched chain substituted O-alkylhydrazide, C₂ -C₅₀ branched chain substituted S-alkylhydrazide, C₂ -C₅₀ branched chain substituted NH-alkylhydrazide, C₂ -C₂₀ straight chain alkenylhydrazide, C₂ -C₂₀ straight chain O-alkenylhydrazide, C₂ -C₂₀ straight chain S-alkenylhydrazide, C₂ -C₂₀ straight chain NH-alkenylhydrazide, C₂ -C₂₀ straight chain substituted alkenylhydrazide, C₂ -C₂₀ straight chain substituted O-alkenylhydrazide, C₂ -C₂₀ straight chain substituted S-alkenylhydrazide, C₂ -C₂₀ straight chain substituted NH-alkenylhydrazide, C₃ -C₅₀ branched chain alkenylhydrazide, C₃ -C₅₀ branched chain O-alkenylhydrazide, C₃ -C₅₀ branched chain S-alkenylhydrazide, C₃ -C₅₀ branched chain NH-alkenylhydrazide, C₃ -C₅₀ branched chain substituted alkenylhydrazide, C₃ -C₅₀ branched chain substituted O-alkenylhydrazide, C₃ -C₅₀ branched chain substituted S-alkenylhydrazide, C₃ -C₅₀ branched chain substituted NH-alkenylhydrazide, C₂ -C₂₀ straight chain alkynylhydrazide, C₂ -C₂₀ straight chain O-alkynylhydrazide, C₂ -C₂₀ straight chain S-alkynylhydrazide, C₂ -C₂₀ straight chain NH-alkynylhydrazide, C₂ -C₂₀ straight chain substituted alkynylhydrazide, C₂ -C₂₀ straight chain substituted O-alkynylhydrazide, C₂ -C₂₀ straight chain substituted S-alkynylhydrazide, C₂ -C₂₀ straight chain substituted NH- alkynylhydrazide, C₃ -C₅₀ branched chain alkynylhydrazide, C₂ -C₅₀ branched chain O-alkynylhydrazide, C₃ -C₅₀ branched chain S-alkynylhydrazide, C₃ -C₅₀ branched chain NH-alkynylhydrazide, C₃ -C₅₀ branched chain substituted alkynylhydrazide, C₃ -C₅₀ branched chain substituted O-alkynylhydrazide, C₃ -C₅₀ branched chain substituted S-alkynylhydrazide, C₃ -C₅₀ branched chain substituted NH-alkynylhydrazide, C₁ -C₂₀ straight chain alkanol, C₁ -C₂₀ straight chain O-alkanol, C₁ -C₂₀ straight chain S-alkanol, C₁ -C₂₀ straight chain NH-alkanol, C₁ -C₂₀ straight chain substituted alkanol, C₁ -C₂₀ straight chain substituted O-alkanol, C₁ -C₂₀ straight chain substituted S-alkanol, C₁ -C₂₀ straight chain substituted NH-alkanol, C₂ -C₅₀ branched chain alkanol, C₂ -C₅₀ branched chain O-alkanol, C₂ -C₅₀ branched chain S-alkanol, C₂ -C₅₀ branched chain NH-alkanol, C₂ -C₅₀ branched chain substituted alkanol, C₂ -C₅₀ branched chain substituted O-alkanol, C₂ -C₅₀ branched chain substituted S-alkanol, C₂ -C₅₀ branched chain substituted NH-alkanol, C₂ -C₂₀ straight chain alkenol, C₂ -C₂₀ straight chain O-alkenol, C₂ -C₂₀ straight chain S-alkenol, C₂ -C₂₀ straight chain NH-alkenol, C₂ -C₂₀ straight chain substituted alkenol, C₂ -C₂₀ straight chain substituted O-alkenol, C₂ -C₂₀ straight chain substituted S-alkenol, C₂ -C₂₀ straight chain substituted NH-alkenol, C₃ -C₅₀ branched chain alkenol, C₃ -C₅₀ branched chain O-alkenol, C₃ -C₅₀ branched chain S-alkenol, C₃ -C₅₀ branched chain NH-alkenol, C₃ -C₅₀ branched chain substituted alkenol, C₃ -C₅₀ branched chain substituted O-alkenol, C₃ -C₅₀ branched chain substituted S-alkenol, C₃ -C₅₀ branched chain substituted NH-alkenol, C₂ -C₂₀ straight chain alkynol, C₂ -C₂₀ straight chain O-alkynol, C₂ -C₂₀ straight chain S-alkynol, C₂ -C₂₀ straight chain NH-alkynol, C₂ -C₂₀ straight chain substituted alkynol, C₂ -C₂₀ straight chain substituted O-alkynol, C₂ -C₂₀ straight chain substituted S-alkynol, C₂ -C₂₀ straight chain substituted NH-alkynol, C₃ -C₅₀ branched chain alkynol, C₂ -C₅₀ branched chain O-alkynol, C₃ -C₅₀ branched chain S-alkynol, C₃ -C₅₀ branched chain NH-alkynol, C₃ -C₅₀ branched chain substituted alkynol, C₃ -C₅₀ branched chain substituted O-alkynol, C₃ -C₅₀ branched chain substituted S-alkynol, C₃ -C₅₀ branched chain substituted NH-alkynol, C₁ -C₂₀ straight chain alkanthiol, C₁ -C₂₀ straight chain O-alkanthiol, C₁ -C₂₀ straight chain S-alkanthiol, C₁ -C₂₀ straight chain NH-alkanthiol, C₁ -C₂₀ straight chain substituted alkanthiol, C₁ -C₂₀ straight chain substituted O-alkanthiol, C₁ -C₂₀ straight chain substituted S-alkanthiol, C₁ -C₂₀ straight chain substituted NH-alkanthiol, C₂ -C₅₀ branched chain alkanthiol, C₂ -C₅₀ branched chain O-alkanthiol, C₂ -C₅₀ branched chain S-alkanthiol, C₂ -C₅₀ branched chain NH-alkanthiol, C₂ -C₅₀ branched chain substituted alkanthiol, C₂ -C₅₀ branched chain substituted O-alkanthiol, C₂ -C₅₀ branched chain substituted S-alkanthiol, C₂ -C₅₀ branched chain substituted NH-alkanthiol, C₂ -C₂₀ straight chain alkenthiol, C₂ -C₂₀ straight chain O-alkenthiol, C₂ -C₂₀ straight chain S-alkenthiol, C₂ -C₂₀ straight chain NH-alkenthiol, C₂ -C₂₀ straight chain substituted alkenthiol, C₂ -C₂₀ straight chain substituted O-alkenthiol, C₂ -C₂₀ straight chain substituted S-alkenthiol, C₂ -C₂₀ straight chain substituted NH-alkenthiol, C₃ -C₅₀ branched chain alkenthiol, C₃ -C₅₀ branched chain O-alkenthiol, C₃ -C₅₀ branched chain S-alkenthiol, C₃ -C₅₀ branched chain NH-alkenthiol, C₃ -C₅₀ branched chain substituted alkenthiol, C₃ -C₅₀ branched chain substituted O-alkenthiol, C₃ -C₅₀ branched chain substituted S-alkenthiol, C₃ -C₅₀ branched chain substituted NH-alkenthiol, C₂ -C₂₀ straight chain alkynthiol, C₂ -C₂₀ straight chain O-alkynthiol, C₂ -C₂₀ straight chain S-alkynthiol, C₂ -C₂₀ straight chain NH-alkynthiol, C₂ -C₂₀ straight chain substituted alkynthiol, C₂ -C₂₀ straight chain substituted O-alkynthiol, C₂ -C₂₀ straight chain substituted S-alkynthiol, C₂ -C₂₀ straight chain substituted NH-alkynthiol, C₃ -C₅₀ branched chain alkynthiol, C₂ -C₅₀ branched chain O-alkynthiol, C₃ -C₅₀ branched chain S-alkynthiol, C₃ -C₅₀ branched chain NH-alkynthiol, C₃ -C₅₀ branched chain substituted alkynthiol, C₃ -C₅₀ branched chain substituted O-alkynthiol, C₃ -C₅₀ branched chain substituted S-alkynthiol and C₃ a-C₅₀ branched chain substituted NH-alkynthiol.

The present invention also provides oligonucleotide cross-linkers for connecting two oligonucleotide strands or for connecting two separate regions of a single strand. In certain embodiments, the cross-linkers are of a structure:

    X.sub.1 --L.sub.1 --Y--L.sub.2 --X.sub.2

wherein X₁ is covalently connected to the first strand or to a first region of a single strand and X₂ is covalently connected to the second strand or to a further region of a single strand and where:

(i) X₁ and X₂, independently, are O, S, NH, CH₂ or CO;

L₁ and L₂, independently, are C₁ -C₂₀ straight chain alkyl, C₁ -C₂₀ straight chain substituted alkyl, C₂ -C₅₀ branched chain alkyl, C₂ -C₅₀ branched chain substituted alkyl, C₂ -C₂₀ straight chain alkenyl, C₂ -₂₀ straight chain substituted alkenyl, C₃ -C₅₀ branched chain alkenyl, C₃ -C₅₀ branched chain substituted alkenyl, C₂ -C₂₀ straight chain alkyne, C₂ -C₂₀ straight chain substituted alkyne, C₃ -C₅₀ branched chain alkyne, C₃ -C₅₀ branched chain substituted alkyne, polyamine, polyamide, polyester, polyethylene glycol, polyether, aryl, aralkyl or heterocyclic; and

Y is an imine, amine, oxime, hydroxylamine, hydrazine, hydrazone, azine, hydrazide-hydrazone, amide, hydrazide, semicarbazide, semicarbazone, thiosemicarbazide, thiocarbazone, disulfide, hemiacetal, thiohemiacetal, α-keto-alkylthioalkyl or αketo-alkylaminoalkyl; or

(ii) X₁ and X₂, independently, are O or NH; and

L₁ --Y--L₂ together are NH--L₃ --NH or NH--NH--L₃ --NH--NH; and

L₃ is C₁ -C₂₀ straight chain alkyl, C₁ -C₂₀ straight chain substituted alkyl, C₂ -C₅₀ branched chain alkyl, C₂ -C₅₀ branched chain substituted alkyl, C₂ -C₂₀ straight chain alkenyl, C₂ -C₂₀ straight chain substituted alkenyl, C₃ -C₅₀ branched chain alkenyl, C₃ -C₅₀ branched chain substituted alkenyl, C₂ -C₂₀ straight chain alkyne, C₂ -C₂₀ straight chain substituted alkyne, C₃ -C₅₀ branched chain alkyne, C₃ -C₅₀ branched chain substituted alkyne, polyamine, polyamide, polyester, polyethylene glycol, polyether, aryl, aralkyl or heterocyclic; or

(iii) X₁ and X₂, independently, are O, S, NH, CH₂ or CO;

L₁ and L₂, independently, are C₁ -C₂₀ straight chain alkylamino, C₁ -C₂₀ straight chain substituted alkylamino, C₂ -C₅₀ branched chain alkylamino, C₂ -C₅₀ branched chain substituted alkylamino, C₂ -C₂₀ straight chain alkenylamino, C₂ -C₂₀ straight chain substituted alkenylamino, C₃ 14 C₅₀ branched chain alkenylamino, C₃ -C₅₀ branched chain substituted alkenylamino, C₂ -C₂₀ straight chain alkynylamino, C₂ -C₂₀ straight chain substituted alkynylamino, C₃ -C₅₀ branched chain alkynylamino, C₃ -C₅₀ branched chain substituted alkynylamino, C₁ -C₂₀ straight chain alkylthio, C₁ -C₂₀ straight chain substituted alkylthio, C₂ -C₅₀ branched chain alkylthio, C₂ -C₅₀ branched chain substituted alkylthio, C₂ -C₂₀ straight chain alkenylthio, C₂ -C₂₀ straight chain substituted alkenylthio, C₃ -C₅₀ branched chain alkenylthio, C₃ -C₅₀ branched chain substituted alkenylthio, C₂ -C₂₀ straight chain alkynylthio, C₂ -C₂₀ straight chain substituted alkynylthio, C₃ -C₅₀ branched chain alkynylthio or C₃ -C₅₀ branched chain substituted alkynylthio; and

Y is a heterobifunctional or homobifunctional cross-linking reagent;

where said substituent of said substituted alkyl, substituted alkenyl and substituted alkyne moieties is an intercalator, a conjugate, polyamine, polyamide, polyethylene glycol, a group that enhances the pharmacodynamic properties of oligonucleotides or a group that enhances the pharmacokinetic properties of oligonucleotides.

As illustrated in the examples below, the invention further includes methods for fixing a sequence-specific oligonucleotide in a set configuration. These methods include selecting a first site in the oligonucleotide. The first site is selected to have a first specific sequence of nucleotides. A first bond precursor is attached to a sugar moiety of one of the nucleotides of the first site. The first bond precursor is for forming a covalent bond. The methods include selecting a second site in the oligonucleotide. The second site is selected to have a second specific nucleotide sequence. A second bond precursor is attached to a sugar moiety of a nucleotide of the second site. The second bond precursor also is for forming a covalent bond. The methods further include forming a covalent cross-linkage between the first and the second bond precursors. The covalent cross-linkage is independent of the phosphate inter-nucleoside linkages that define-the primary structure of the oligonucleotide. In certain embodiments, the methods further include attaching the first bond precursor to a sugar moiety of a nucleotide of the first site by reacting a precursor of the nucleotide with a suitable reagent, derivatizing the nucleotide precursor to a protected and activated form, and then synthetically incorporating the derivatized nucleotide precursor into the first nucleotide sequence. In still other embodiments, the methods further include attaching the second bond precursor to a sugar moiety of a nucleotide of the second site by reacting a precursor of the nucleotide with a suitable reagent, derivatizing the nucleotide precursor to a protected and activated form, and synthetically incorporating the derivatized precursor into the second nucleotide sequence.

The invention further includes methods for the intrastrand cross-linkage of an oligonucleotide. In certain embodiments, the methods include the steps of selecting a first nucleotide sequence and a second nucleotide sequence in the oligonucleotide, incorporating an abasic nucleotide within the first sequence, attaching a bond precursor to a nucleotide of the second sequence, and forming a covalent cross-linkage between the abasic site and the bond precursor.

In other embodiments, the methods include selecting a first nucleotide sequence and a second nucleotide sequence in the oligonucleotide, attaching a first bond precursor to a sugar moiety of a nucleotide of the first specific sequence, attaching a second bond precursor to a sugar moiety of a nucleotide of the second sequence, and forming covalent cross-linkage between the first and the second bond precursors. This covalent cross-linkage is independent of the phosphate inter-nucleotide linkages that define the primary structure of the oligonucleotide. Preferably, the first nucleotide sequence is selected to be complementary to and hybridizable with the second nucleotide sequence.

The invention further includes methods for cross-linking first and second oligonucleotide strands including the steps of selecting a first nucleotide sequence in a first oligonucleotide strand, selecting a second nucleotide sequence in a second oligonucleotide strand, incorporating an abasic nucleotide within the first nucleotide sequence, attaching a bond precursor to a nucleotide of the second sequence, and forming a covalent cross-linkage between the abasic site and the bond precursor. These methods can further include selecting the first nucleotide sequence to be complementary to and hybridizable with the second nucleotide sequence, attaching the bond precursor at a 2'-position of a nucleotide of the second sequence, and forming an aldehydic functional group at the abasic site. The bond precursor should be capable of forming an oxime, imine, hydrazone, semicarbazone, thiosemicarbazone, hydrazide-hydrazone, hemiacetal or thiohemiacetal linking group with the abasic site aldehydic functional group.

The invention further includes methods for cross-linking first and second oligonucleotide strands including the steps of selecting a first nucleotide sequence in a first oligonucleotide strand, selecting a second nucleotide sequence in a second oligonucleotide strand, attaching a first bond precursor to a sugar moiety of a nucleotide of the first sequence, attaching a bond precursor on a sugar moiety of a nucleotide of the second sequence, and forming a covalent cross-linkage between the first and the second bond precursors. This covalent cross-linkage is independent of the phosphate inter-nucleotide linkages that define oligonucleotide primary structure.

The following examples are illustrative of the invention. It is understood that this invention is not limited by these illustrative examples but solely by the claims appended hereto.

EXAMPLE 1

Standard Oligonucleotide Synthesis

Oligonucleotide syntheses were performed on an Applied Biosystems 380 B or 394 DNA synthesizer following standard phosphoramidite protocols and cycles using reagents supplied by the manufacture. When modified phosphoramidites are used, a longer coupling time (10-15 min) was employed. The oligonucleotides were normally synthesized in either a 10 μmol scale or a 3×1 μmol scale in the "Trityl-On" mode. Standard deprotection conditions (30% NH₄ OH, 55° C., 16 hr) were employed. HPLC was performed on a Waters 600E instrument equipped with a model 991 detector. For analytical chromatography, the following reverse phase HPLC conditions were employed: Hamilton PRP-1 column (15×2.5 cm); solvent A: 50 mm TEAA, pH 7.0; solvent B: 45 mm TEAA with 80% CH₃ CN; flow rate: 1.5 ml/min; gradient: 5% B for the first 5 minutes, linear (1%) increase in B every minute thereafter. For preparative purposes, the following reverse phase HPLC conditions were employed: Waters Delta Pak Waters Delta-Pak C₄ 15 μm, 300A, 25×100 mm column equipped with a guard column of the same material; column flow rate: 5 ml/min; gradient: 5% B for the first 10 minutes, linear 1% increase for every minute thereafter. Following HPLC purification, oligonucleotides are detritylated and further purified by size exclusion using a Sephadex G-25 column. All oligonucleotide sequences are listed in a standard 5' to 3' order from left to right.

EXAMPLE 2

Preparation Of Activated and Protected Nucleotides Having A Space-spanning Group And Active Functional Group

A. Preparation of 5'-Dimethoxytrityl-2'-O-(Functionalized Space-spanning Group)Nucleoside Phosphoramidites

i. 5'-Dimethoxytrityl-2'-O-(Pentyl-N-phthalimido)-N6-Benzyladenosine Phosphoramidite

5'-Dimethoxytrityl-2'-O-(pentyl-N-phthalimido)-N6-benzyladenosine phosphoramidite was synthesized as per the procedures of U.S. patent applications Ser. Nos. 91/00243 and 463,358, herein incorporated by reference, starting from adenosine. Briefly, this procedure treats adenosine with NaH in DMF followed by treatment with N-(5-bromopentyl)phthalimide (Transworld Chemical Co. Inc., Rockville, Md.). Further treatment with (CH₃)₃ SiCl, Ph--C(O)--Cl and NH₄ OH yielded N6-benzyl protected 2'-pentyl-N-phthalimido functionalized adenosine. Treatment with DIPA and CH₂ Cl₂ added a DMT blocking group at the 5'-position. Finally phosphitylation gave the desired phosphoramidite compound.

ii. 5'-Dimethoxytrityl-2'-O-(2-Methyloctyl-N-phthalimido)-N4-Benzylcytidine Phosphoramidite

a. N-(8-bromo-2-methyloctyl)phthalimide

8-Bromo-2-methylaminooctane is treated with phthalic anhydride in toluene in the presence of triethylamine under reflux. The solvent is removed in vacuo. The residue, when triturated with water, acidified with hydrochloric acid and crystallized from alcohol will yield N-(8-bromo-2-methyloctyl)phthalimide.

b. Preparation of 5'-Dimethoxytrityl-2'-O-(2-Methyloctyl-N-Phthalimido)-N4-Benzylcytidine Phosphoramidite

Cytidine, when treated with NaH and N-(8-Bromo-2-methyloctyl)phthalimide followed by treatment with DIPA and CH₂ Cl₂ and phosphitylation utilizing the procedure of Example 2-A-i, will give 5'-dimethoxytrityl-2'-O-(2-methyloctyl-N-phthalimido)-N4-benzylcytidine phosphoramidite.

c. 5'-Dimethoxytrityl-2'-O-(Eicosyl-N-Phthalimido)Adenosine Phosphoramidite

N-(20-bromoeicosyl)phthalimide is prepared as per the procedure of Example 2-A-ii-a from 1-amino-20-bromoeicosane. Adenosine is treated with NaH in DMF and 1-amino-20-bromoeicosane, benzylated, treated with DIPA and CH₂ Cl₂ and phosphitylation as per the procedure of Example 2-A-ii will yield the title compound.

iii. 5'-Dimethoxytrityl-2'-O-(Eicosyl-N-Phthalimido)Uridine Phosphoramidite

N-(20-bromoeicosyl)phthalimide is prepared as per the procedure of Example 2-A-ii-a from 1-amino-20-bromoeicosane. Uridine is treated as per the procedure of Akiyama, et al., NOTES Bull. Chem. Soc. Jpn., 1990, 63, 3356, to give 3-(4-methoxyphenylmethyl)-3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)uridine. Briefly this treatment first blocks the 3' and 5' sugar hydroxyls of uridine followed by blocking the N3 imide group of the pyrimidine heterocycle with a p-methoxybenzyl group. Treatment of the blocked uridine with Ag₂ O and N-(20-bromoeicosyl)phthalimide will yield a 2'-alkylated blocked intermediate compound. The tetraisopropyldisiloxane blocking group is removed by taking the uridine intermediate up in CH₃ CN and treating with 60% HF. The p-methoxyphenylmethyl blocking group is removed by treatment with AlCl₃ in anisole under a N₂ atmosphere followed by Hcl. Both of these procedures are conducted as per the above procedure of Akiyama et al. Further treatment as per the procedure of Example 2-A-ii with DIPA and CH₂ Cl₂ to add a DMT blocking group at the 5'-position followed by phosphitylation will yield the title compound.

iv. 5'-Dimethoxytrityl-2'-O-(2-Amylenyl-N-phthalimido)-N2-Isobutyrylguanosine Phosphoramidite

N-(5-bromo-2-amylenyl)phthalimide is prepared as per the procedure of Example 2-A-ii-a from 1-amino-5-bromoamyl-2-ene. 3',5'-O-(Tetraisopropyldisiloxane-1,3-diyl)-2-chloro-6-(2,6-dichlorophenoxy)purine riboside is prepared as described by Sproat, Nucleic Acid Res., 1990, 18, 41. Treatment of the latter compound with the first followed by deblocking and enzymatic deaminization with adenosine deaminase utilizing the procedure of Sproat, Nucleic Acids Research, 1991, 19, 733, will yield the desired 2'-alkylated guanosine intermediate. Further treatment of this alkylated guanosine intermediate as per the procedure of Example 2-A-i with (CH₃)₃ SiCl, isobutyryl chloride and NH₄ OH yields N2-isobutyryl protected 2'-amylenyl-N-phthalimido functionalized guanosine. Treatment with DIPA and CH₂ Cl₂ to add a DMT blocking group at the 5'-position followed by phosphitylation will give the desired phosphoramidite compound.

v. 5'-Dimethoxytrityl-2'-O-(Ethyl-N-Phthalimido)Thymidine Phosphoramidite

Thymidine is blocked and treated with Ag₂ O and N-(2-bromoethyl)phthalimide (Aldrich Chemical Co., Inc., Milwaukee, Wis.) as per the procedure of Example 2-A-iii followed by further treatment as per the procedure of Example 2-A-i with DiPA and CH₂ Cl₂ to add a DMT blocking group at the 5'-position and finally phosphitylation will yield the title compound.

vi. Preparation of Other 5'-Dimethoxytrityl-2'-O-(Phthalimido Blocked Amine Functionalized Space-spanning Group)-Nucleoside Phosphoramidites

a. Preparation of N-(Ω-bromoalkyl, Ω-bromoalkenyl and Ω-bromoalkynyl)phthalimides

In the manner of Example 2-A-ii-a the following Ω-bromo phthalimide blocked amine compounds, useable as amine functionalized space-spanning groups, can be prepared:

N-(7-bromoheptyl)phthalimide;N-(8-bromooctyl)phthalimide; N-(9-bromononyl)phthalimide; N-(10-bromododecyl)phthalimide; N-(11-bromoundecyl)phthalimide; N-(12-bromodocecyl)phthalimide; N-(13-bromotridecyl)phthalimide; N-(14-bromotetradecyl)phthalimide; N-(15-bromopentadecyl)-phthalimide; N-(16-bromohexadecyl)phthalimide; N-(17-bromoheptadecyl)phthalimide;N-(18-bromooctadecyl)phthalimide; N-(19-bromononadecyl)phthalimide; N-(3-bromo-2-methylpropyl)phthalimide; N-(4-bromo-2-methyl-3-ethylbutyl)phthalimide; N-(3-bromo-2,2-diethylpropyl)phthalimide; N-(4-bromo-3-propylbutyl)phthalimide; N-(10-bromo-2,8-dibutyl-decyl)phthalimide; N-(8-bromo-6,6-dimethyloctyl)phthalimide; N-(8-bromo-6-propyl-6-butyloctyl)phthalimide; N-(4-bromo-2-methylbutyl)phthalimide; N-(5-bromo-2-methylpentyl)phthalimide; N-(5-bromo-3-methylpentyl)phthalimide; N-(6-bromo-2-ethylhexyl)phthalimide; N-(5-bromo-3-penten-2-one)phthalimide; N-(4-bromo-3-methyl-2-butanol)phthalimide; N-(8-bromo-3-amino-2-cyanooctyl)phthalimide; N-(7-bromo-3-methoxy-4-heptanal)phthalimide; N-(4-bromo-2-iodo-3-nitrobutyl)phthalimide; N-(12-bromo-4-isopropoxydodecyl)phthalimide; N-(10-bromo-4-azido-2-nitrodecyl)phthalimide; N-(9-bromo-5-mercaptononyl)phthalimide; N-(5-bromo-4-aminopentenyl)phthalimide;N-(5-bromo-penten-2-yl)phthalimide; N-(3-bromoallyl)phthalimide; N-(4-bromocrotyl)phthalimide; N-(3-bromopropargyl)phthalimide; N-(1-bromonaphth-4-yl)phthalimide; N-(2-bromoanthrac-7-yl)phthalimide; and N-(2-bromophenanthr-6-yl)phthalimide.

b. Preparation of 5'-Dimethoxytrityl-2'-O-(N-Phthalimide Functionalized Space-spanning Group)Nucleoside Phosphoramidites

In the manner of Examples 2-A-i, 2-A-ii-b, 2-A-iii, 2-A-iv and 2-A-v, the following 5'-dimethoxytrityl-2'-O-(space-spanning group-N-phthalimide)nucleoside phosphoramidite can be prepared:

5'-dimethoxytrityl-2'-O-(dodecyl-N-phthalimido)-N4-benzylcytidine phosphoramidite; 5'-dimethoxytrityl-2'-O-(tetra-decyl-N-phthalimido)-N6-benzyladenosine phosphoramidite; 5'-dimethoxytrityl-2'-O-(2-methylpropyl-N-phthalimido)uridine phosphoramidite; 5'-dimethoxytrityl-2'-O-(2,8-dibutyldecyl-N-phthalimido)-N2-isobutyrylguanosine phosphoramidite; 5'-dimethoxytrityl-2'-O-(3-methylpentyl-N-phthalimido)thymidine phosphoramidite; 5'-dimethoxytrityl-2'-O-(2-cyanooctyl-N-phthalimido)-N4-benzylcytidine phosphoramidite; 5'-dimethoxy-trityl-2'-O-(3-methoxy-4-heptanal-N-phthalimido)uridine phosphoramidite; 5'-dimethoxytrityl-2'-O-(3-nitrobutyl-N-phthalimido)-N6-adenosine phosphoramidite; 5'-dimethoxytrityl-2'-O-(propargyl-N-phthalimido)-N2-isobutyrylguanosine phosphoramidite; and 5'-dimethoxytrityl-2'-O-(3-anthrac-7-yl-N-phthalimido) thymidine phosphoramidite.

B. Preparation of 5'-Dimethoxytrityl-2'-S-(Amine Functionalized Space-spanning Group)-2'-Deoxy-Nucleoside Phosphoramidites

i. Preparation of N-(1-Bromo-8-dodecyl)Phthalimide

1-Aminododecyl-12-ol is treated with N-ethoxycarbonylphthalimide utilizing the reaction conditions of McArthur, et. al., Synth. Commun., 1983, 13, 311, to yield N-(dodecyl-12-ol)phthalimide which in turn when treated with phosphorous tribromide and pyridine utilizing the reaction conditions of Hall, et al., Synthetic Procedures in Nucleic Acid Chemistry, Zorbach and Tipson, Editors, Volume 1, John Wiley & Sons, 1968 will yield N-(12-bromododecyl)phthalimide.

ii. 5'-Dimethoxytrityl-2'-S -(Dodec-12-N-Phthalimido)-2'-Deoxyuridine Phosphoramidite

2'-Deoxy-2-thiouridine is prepared as per the procedure of Divakar, J. Chem. Soc. Perkin Trans., 1990, 1, 969. 2'-Deoxy-2-thiouridine when treated with NaH in DMF followed by treatment with N-(12-bromododecyl)phthalimide will yield 2'-S-(dodecyl-N-phthalimido) functionalized uridine. Treatment with DIPA and CH₂ Cl₂ to add a DMT blocking group at the 5'-position followed by phosphitylation will give the desired phosphoramidite compound.

C. Preparation of 5'-Dimethoxytrityl-2'-NH-(Alkyl-N-Phthalimido)-2'-Deoxy-Nucleoside Phosphoramidites

i. N-(Butyl-4-al)Phthalimide (an Ω-aldehydic terminating α-N-Phthalimide functionalized space-spanning group)

4-Aminobutyraldehyde diethyl acetal (Aldrich Chemical Co., Inc., Milwaukee, Wis.) when treated with phthalic anhydride in toluene in the presence of triethylamine as per the procedure of Example 2-A-ii-a will give an N-phthalimide blocked amine group at the 1-position of the butyl group and an diethyl acetal blocked aldehyde group at the 4-position. The diethyl acetal blocking group is then removed by acid catalyzed hydrolysis to give N-(butyl-4-al)phthalimide.

ii. 5'-Dimethoxytrityl-2'-NH-(Butyl-N-Phthalimido)-2'-Deoxy-N6-Benzyladenosine Phosphoramidite

2'-Amino-N6-benzyl-2'-deoxyadenosine, prepare as per the procedure of G. Burke, et. al., J. Carbohydrates, Nucleosides, Nucleotides, 1988, 7, 63, and N-(butyl-4-al)phthalimide are stirred in dry CH₂ Cl₂. The solvent is removed and the residue taken up in NaOAc buffer (pH 5.02) and treated with NaCNBH₃ to reduce the intermediate imine to the desired amine linking functionality. Further treatment as per the procedure of Example 2-A-i with DIPA and CH₂ Cl₂ to add a DMT blocking group at the 5'-position followed by phosphitylation will yield the title compound.

D. Preparation of 5'-Dimethoxytrityl-2'-(Alkyl-N-Phthalimido)-2'-Deoxy-Nucleoside Phosphoramidites

Introduction of a 2' directly alkylated nucleotide (i.e. a methylene linking group joining a space-spanning group to the 2'-position of a nucleotide sugar moiety) into an oligonucleotide is accomplished by free radical alkylation of the corresponding nucleoside followed by conversion to the appropriate 5'-dimethoxytrityl phosphoramidite nucleotides.

i. 5'-Dimethoxytrityl-2'-(Allyl-N-Phthalimido)-2'-Deoxy-N6-Benzyladenosine Phosphoramidite

2'-O-(Phenoxythioxomethyl)-2'-deoxyadenosine will be treated with N-(5-bromopenten-2-yl)phthalimide (from Example 2-A-vi) and tributyltinhydride utilizing conditions analogous to those described in Flandor, et al., Tetrahedron Letters, 1990, 31, 597, to effect a free radical alkylation at the 2'-position of the nucleoside. The resulting alkylated compound is then further treated with (CH₃)₃ SiCl, Ph--C(O)--Cl and NH₄ OH to give the N6-benzyl protected 2'- N-(penten-2-yl)phthalimido! functionalized adenosine. This is then followed by treatment with DIPA and CH₂ Cl₂ to add a DMT blocking group at the 5'-position which in turn is followed by phosphitylation, all as per the procedure of Example 2-A-i, to give the desired phosphoramidite compound.

ii. 5'-Dimethoxytrityl-2'-(Allyl-N-Phthalimido)-2'-Deoxyuridine Phosphoramidite

2'-O-(Phenoxythioxomethyl)-2'-deoxyuridine will be treated with N-(5-bromopenten-2-yl)phthalimide (from Example 2-A-vi) and tributyltinhydride utilizing conditions analogous to those described in Flandor, et al., Tetrahedron Letters, 1990, 31, 597 to effect a free radical alkylation at the 2'-position of the nucleoside. The resulting alkylated compound is then further treated with DIPA and CH₂ Cl₂ to add a DMT blocking group at the 5'-position which in turn is followed by phosphitylation, all as per the procedure of Example 2-A-iii, to give the desired phosphoramidite compound.

E. Preparation of 5'-Dimethoxytrityl-2'-O-(Hydrazine Functionalized Space-spanning Group)Nucleoside Phosphoramidites

i. 8-Bromooctylbenzylcarbazide (an α-bromo carbazide protected Ω-hydrazine functionalized space-spanning group)

8-Bromooctanol is tosylated with tosyl chloride in pyridine. The solvent is removed and the 8-bromooctanol O-tosylate is treated with benzylcarbazide and activated molecular sieves in anhydrous dimethylacetamide overnight at 110° C. The solvent is removed and the residue purified by silica gel chromatography to yield bromooct-8-ylbenzylcarbazide.

ii. 5'-Dimethoxytrityl-2'-O-(Oct-8-ylbenzylcarbazide)uridine Phosphoramidite

Uridine will be treated with 8-bromooctylbenzylcarbazide utilizing the method of Example 2-A-iii to give the title compound.

iii. 5'-Dimethoxytrityl-2'-O-(Oct-8-ylbenzyl-carbazide)-N4-Benzylcytidine Phosphoramidite

Cytidine will be treated with 8-bromooctylbenzylcarbazide utilizing the method of Example 2-A-ii-b to give the title compound.

iv. 5'-Dimethoxytrityl-2'-O-(Oct-8-ylbenzylcarbazide)-N2-Isobutyrylguanosine Phosphoramidite

Guanosine will be treated with 8-bromooctylbenzylcarbazide utilizing the method of Example 2-A-iv to give the title compound.

v. 5'-Dimethoxytrityl-2'-O-(Oct-8-ylbenzylcarbazide)-N6-Benzyladenosine Phosphoramidite

Adenosine will be treated with 8-bromooctylbenzylcarbazide utilizing the method of Example 2-A-i to give the title compound.

F. Preparation of 5'-Dimethoxytrityl-2'-O-(Hydroxylamine-Functionalized Space-spanning Group)Nucleoside Phosphoramidites

i. O-(8-Bromooctanol)phthalimide (an α-bromo O-phthalimide protected Ω-hydroxylamine functionalized space-spanning group)

1,8-Octanediol is treated with one equivalent of dihydropyrane in the presence of acid to give the 8-(tetrahydropyran-2-yl)octanol. 8-(Tetrahydropyran-2-yl)octanol, N-hydroxyphthalimide and triphenylphosphine in DMF are treated at 0° C. with diisopropylazodicarboxylate utilizing Mitsunobu reaction conditions to give O- 8-(tetrahydropyran-2-yl)octanol)phthalimide. Acid treatment of O- 8-(tetrahydropyran-2-yl)octanol!phthalimide will yield O-(octanol)phthalimide which in turn when treated with phosphorous tribromide and pyridine utilizing the reaction conditions of Hall, et al., Synthetic Procedures in Nucleic Acid Chemistry, Zorbach and Tipson, Editors, Volume 1, John Wiley & Sons, 1968 will yield O-(8-bromooctanol)phthalimide.

ii. 5'-Dimethoxytrityl-2'-O-(Octan-8-ol-O-Phthalimido)-N4-Benzylcytidine Phosphoramidite

Cytidine is treated with NaH in DMF followed by treatment with O-(8-bromooctanol)phthalimide from Example 2-F-i. Further treatment with (CH₃)₃ SiCl, Ph--C(O)--Cl and NH₄ OH will yield N4-benzyl protected 2'-octyl-O-phthalimido functionalized cytidine. Treatment with DIPA and CH₂ Cl₂ will add a DMT blocking group at the 5'-position. Finally phosphitylation as per the procedure of Example 2-A-ii will give the desired phosphoramidite compound.

iii. 5'-Dimethoxytrityl-2'-O-(Octan-8-ol-O-Phthalimide)uridine Phosphoramidite

Uridine is blocked and then treated with Ag₂ O and O-(8-bromooctanol)phthalimide from Example 2-F-i. Treatment with DIPA and CH₂ Cl₂ will add a DMT blocking group at the 5'-position. Finally phosphitylation as per the procedure of Example 2-A-iii will give the desired phosphoramidite compound.

G. Preparation of 5'-Dimethoxytrityl-2'-O-(Semicarbazide Functionalized Space-spanning Group)Nucleoside Phosphoramidites

i. 5'-Dimethoxytrityl-2'-O-(pent-5-ylsemicarbazide)uridine Phosphoramidite

Uridine is blocked and then treated with Ag₂ O and N-(8-bromopenyl)semicarbazide utilizing the method of Example 2-A-iii to give the title compound.

ii. 5'-Dimethoxytrityl-2'-O-(pent-5-ylsemicarbazide)-N6-Benzyladenosine Phosphoramidite

Adenosine will be treated with N-(8-bromopenyl)semicarbazide utilizing the method of Example 2-A-i to give the title compound.

H. Preparation of 5'-Dimethoxytrityl-2'-O-(Hydrazide Functionalized Space-spanning Group)Nucleoside Phosphoramidites

i. αCloroethoxycarbonylethane

Chloroacetyl chloride (Air Products, Emmaus, Pa.) is added drop wise to stirring ethanol to esterify the acid chloride. The ester is then recovered by distillation.

ii. 5'-Dimethoxytrityl-2'-O-(ethoxycarbonylethyl)uridine Phosphoramidite

Uridine is blocked and then treated with Ag₂ O and α-cloroethoxycarbonylethane as per the procedure of Example 2-A-iii to yield the title compound.

iii. 5'-Dimethoxytrityl-2'-O-(eth-2-ylhydrazide)uridine Phosphoramidite

5'-Dimethoxytrityl-2'-O-(ethoxycarbonylethyl)uridine phosphoramidite will be treated with hydrazine to give the corresponding 2'-O-(ethylhydrazide)uridine title compound.

I. Preparation of 5'-Dimethoxytrityl-2'-O-(Alcohol Functionalized Space-spanning Group)Nucleoside Phosphoramidites

i. 1-O-(Tetrahydropyran-2-yl)-7-bromoheptanol 7-Bromoheptanol will be treated with dihydropyrane in the presence of acid to give 1-O-(tetrahydropyran-2-yl)-7-bromoheptanol.

ii. 5'-Dimethoxytrityl-2'-O- O-(Tetrahydropyran-2-yl)heptan-7-ol)!-N4-Benzylcytidine Phosphoramidite

Cytidine will be treated with 1-O-(tetrahydropyran-2-yl)-7-bromoheptanol as per the procedure of Example 2-A-ii to give the title compound.

iii. 5'-Dimethoxytrityl-2'-O- O-(Tetrahydropyran-2-yl)heptan-7-ol)!uridine Phosphoramidite

Uridine is blocked and then treated with Ag₂ O and 1-O-(tetrahydropyran-2-yl)-8-bromoheptanol as per the procedure of Example 2-A-iii to give the title compound.

J. Preparation of 5'-Dimethoxytrityl-2'-O or 2'-S (Thiol Functionalized Space-spanning Group)-Nucleoside Phosphoramidites

i. Via Ring Opening of 2,2'-Anhydro Pyrimidines

a. 2'-S-(Octan-8-Thiol) thymidine

2,2'-Anhydro thymidine will be ring opened with octane-1,8-dithiol to give 2'-S-(octan-8-thiol)thymidine.

b. 5'-Dimethoxytrityl-2'-S- S-Trityl(Octan-8-thiol)!-2'-Deoxy-Thymidine Phosphoramidite

2'-S-(octan-8-thiol)thymidine when treated with trityl chloride will give 2'-S- S-trityl(octan-8-thiol)!thymidine which will then be further treated with DIPA and CH₂ Cl₂ to add a DMT blocking group at the 5'-position and finally phosphitylation as per Example 2-A-v to give the title compound.

ii. Via O-Alkylation

a. 1-S-Trityl-8-Bromohexanethiol

8-Bromohexanethiol will be tritylated with trityl chloride to give 1-S-trityl-8-bromohexanethiol.

b. 5'-Dimethoxytrityl-2'-O- S-Trityl-(Hexan-8-thiol)!-N6-Benzyladenosine Phosphoramidite

Adenosine is treated with NaH in DMF as per example 2-A-i. The reaction product will then be treated with 1-S-trityl-8-bromohexanethiol followed by treatment with (CH₃)₃ SiCl, Ph--C(O)--Cl and NH₄ OH to yield the N6-benzyl protected 2'-O- S-trityl(hexyl-8-thiol)! functionalized adenosine. Treatment with DIPA and CH₂ Cl₂ to add a DMT blocking group at the 5'-position followed by phosphitylation, further as per the procedure of Example 2-A-i, will give the title compound.

c. 5'-Dimethoxytrityl-2'-O- S-Trityl-(Hexan-8-thiol)!uridine Phosphoramidite

Uridine is blocked and then treated with Ag₂ O and 1-S-trityl-8'-bromohexanethiol as per Example 2-A-iii. The reaction product will then be treated with followed by treatment with DIPA and CH₂ Cl₂ to add a DMT blocking group at the 5'-position and finally phosphitylation, further as per the procedure of Example 2-A-iii, to give the title compound.

d. 1-S-Trityl-8-Bromopentanethiol

8-Bromopentanethiol will be tritylated with trityl chloride to give 1-S-trityl-8-bromopentanethiol.

e. 5'-Dimethoxytrityl-2'-O- S-Trityl-(Pentan-8-thiol)!uridine Phosphoramidite

Uridine is blocked and then treated with Ag₂ O and 1-S-trityl-8-bromopentanethiol as per Example 2-A-iii followed by treatment with DIPA and CH₂ Cl₂ to add a DMT blocking group at the 5'-position and finally phosphitylation, further as per the procedure of Example 2-A-iii, to give the title compound.

K. Preparation of 5'-Dimethoxytrityl-2'-O-(Protected Aldehyde Functionalized Space-spanning Group)Nucleoside Phosphoramidites

i. Preparation of Acetal Functionalization

a. N6-Benzyl-5'-Dimethoxytrityl-2'-O- Propion-3-al Bis(o-Nitrobenzyl Acetal)adenosine Phosphoramidite

Adenosine is treated with NaH and 3-bromopropionaldehyde bis(o-nitrobenzyl) acetal as per the procedure of Example 2-A-i. Further treatment with (CH₃)₃ SiCl, Ph--C(O)--Cl and NH₄ OH to add a benzyl blocking group followed by DIPA and CH₂ Cl₂ to add a DMT blocking group at the 5'-position and further followed by phosphitylation as per the procedure of Example 2-A-i, will give the desired compound.

b. 5'-Dimethoxytrityl-2'-O- Propion-3-al Bis(o-Nitrobenzyl Acetal)uridine Phosphoramidite

Uridine is blocked followed by treatment with Ag₂ O and 3-bromopropionaldehyde bis(o-nitrobenzyl) acetal as per the procedure of Example 2-A-iii. Further treatment with DIPA and CH₂ Cl₂ to add a DMT blocking group at the 5'-position and further followed by phosphitylation as per the procedure of Example 2-A-i, will give the desired compound.

ii 6-(Diphenylimidazolidino)hexyl Bromide

6-Bromohexionyl acid chloride will be treated with ethylmercaptan. The resulting ethyl thiol-6-bromohexionate is then desulfurized with Raney nickel giving 6-bromohexenal. The 6-bromohexenal is then treated with 1,2-dianilinoethylene in benzene/DMSO to give the title 6-(diphenylimidazolidino)hexyl bromide.

iii. 5'-Dimethoxytrityl-2'-O- 6-(diphenylimidazolidino)hexyl!-N2-Isobutyrylguanosine Phosphoramidite

3',5'-O-(Tetraisopropyldisiloxane-1,3-diyl)-2-chloro-6-(2,6-dichlorophenoxy)purine riboside is reacted with 6-(diphenylimidazolidino)hexyl bromide as per Example 2-A-iv. Further treatment with (CH₃)₃ SiCl, isobutyryl chloride and NH₄ OH will yield N2-isobutyryl protected 2'-(diphenylimidazolidino)hexyl functionalized guanosine. Treatment with DIPA and CH₂ Cl₂ to add a DMT blocking group at the 5'-position followed by phosphitylation, further as per the procedure of Example 2-A-iv, will give the title compound.

iv. 5'-Dimethoxytrityl-2'-O- 6-(diphenylimidazolidino)hexyl!-N6-Benzyladenosine Phosphoramidite.

Adenosine is treated with NaH/DMF followed by treatment with 6-(diphenylimidazolidino)hexyl bromide as per Example 2-A-i. Further treatment with (CH₃)₃ SiCl, benzyl chloride and NH₄ OH will yield N6-benzyl protected 2'-(diphenylimidazolidino)hexyl functionalized adenosine. Treatment with DIPA and CH₂ Cl₂ to add a DMT blocking group at the 5'-position followed by phosphitylation, further as per the procedure of Example 2-A-i, will give the title compound.

v. 5'-Dimethoxytrityl-2'-O- 6-(diphenylimidazolidino)hexyl)-N4-Benzylcytidine Phosphoramidite

Cytidine is treated with NaH/DMF followed by treatment with 6-(diphenylimidazolidino)hexyl bromide as per Example 2-A-ii. Further treatment with (CH₃)₃ SiCl, benzyl chloride and NH₄ OH will yield N4-benzyl protected 2'-(diphenylimidazolidino)hexyl functionalized cytidine. Treatment with DIPA and CH₂ Cl₂ to add a DMT blocking group at the 5'-position followed by phosphitylation, further as per the procedure of Example 2-A-ii, will give the title compound.

vi. 5'-Dimethoxytrityl-2'-O- 6-(diphenylimidazolidino)hexyl!uridine Phosphoramidite

Uridine is blocked and then treated with Ag2O and 6-(diphenylimidazolidino)hexyl bromide as per Example 2-A-iii. Treatment with DIPA and CH₂ Cl₂ to add a DMT blocking group at the 5'-position followed by phosphitylation, further as per the procedure of Example 2-A-ii, will give the titled compound.

EXAMPLE 3

Preparation Of Oligonucleotides Having An Abasic Sites Located Thereon

A. Preparation of An Abasic Site Containing Oligonucleotide Via Enzymatic Reaction

i. Oligonucleotide Containing Single Uridine Site

An oligonucleotide of the sequence:

Oligomer 1 CGC AGU* CAG CC SEQ ID NO:1

wherein U* represents a 2'-deoxy uridine nucleotide, was prepared utilizing the procedure of Example 1. The deoxyuridine nucleotide in the middle of the sequence was added during the synthesis utilizing deoxyuridine phosphoramidite (Glen Research, Sterling, Va.). The oligonucleotide was prepared utilizing standard synthesis cycles. It was deprotected by normal deprotection utilizing ammonium hydroxide, 30%, for 16 hours. The solvent was evaporated, and the residue was purified by HPLC and detritylated. Final purification was effected on Sephadex G-25.

ii. Enzyme Stock Solution Preparation

Uracil-DNA glycosylase was isolated from E. coli M5219 cells transformed with the expression plasmid pBD396 containing the ung gene. The enzyme was purified to electrophoretic homogeneity as described by Lindahl, et al., J. Biol. Chem., 1977, 252, 3286, and stored in 30 Mm HEPES-NaOH, pH 7.4, containing 5% glycerol, 2 mM DTT and 1 mM EDTA.

iii. Oligonucleotide Containing Single Abasic Site

An abasic oligonucleotide of the sequence:

Oligomer 2 CGC AGD* CAG CC SEQ ID NO:2

wherein D* represents an abasic site, was prepared by treating 237 O.D. units of Oligomer 1 of Example 3-A-i in 0.5 ml water with 200 μl of the stock solution of Example 3-A-ii (200 micrograms of uracil DNA-glycosylase) and incubating at room temperature overnight. HPLC analysis showed quantitative removal of uracil as indicated by a 1:10 ratio between uracil and the abasic dodecamer oligonucleotide. The uracil retention time was 2.43 mins and the abasic oligonucleotide was 21.68 mins. The solution was lyophilized and stored in the freezer until further use.

iv. Oligonucleotide Containing Multiple Uridine Sites

In the manner of Example 3-A-i the following oligonucleotide was prepared:

Oligomer 3 GAC AGA GGU* AGG AGA AGU* GA SEQ ID NO:3

wherein U* represents a 2'-deoxyuridine nucleotide. The oligonucleotide when treated as per the procedure of Example 3-A-iii will give an oligonucleotide of the sequence

Oligomer 4 GAC AGA GGD* AGG AGA AGD* GA SEQ ID NO:4

wherein D* represents an abasic site within the oligonucleotide.

B. Preparation of An Abasic Site Containing Oligonucleotide Via Abasic Sugar Precursor

i. 5-O-4,4'-Dimethoxytrityl-1,2-Dideoxy-D-Ribofuranose-3-O-(2-Cyanoethyl-N,N'-Diisopropyl) Phosphoramidite

5-O-4,4'-Dimethoxytrityl-1,2-dideoxy-D-ribofuranose-3-O-(2-cyanoethyl-N,N'-diisopropyl) phosphoramidite is prepared as per the procedure of Lyer, et al., Nucleic Acids Research, 1990, 18, 2855, or as per the procedure of Didier Peoch et al., Tetrahedron Letters, 1991, 32, 207.

ii. Oligonucleotide Containing Abasic Site

Oligomer 2 of Example 3-A-iii, i.e. the oligonucleotide of the sequence:

Oligomer 2 CGC AGD* CAG CC

wherein D* represents an abasic site, can also be prepared utilizing the synthetic procedures of the papers identified in Example 3-B-i. Utilizing those procedures, an o-Nitrobenzyldeoxyfuranose containing oligonucleotide is synthesized using the oligonucleotide synthetic methods of these papers. Photolysis, utilizing a high intensity Hg lamp, generates the corresponding abasic site containing oligonucleotide. Such abasic oligonucleotides are also described by Horn, et al., Nucleosides & Nucleotides, 1991, 10, 299.

EXAMPLE 4

Preparation of Oligonucleotides Incorporating 2'-O-(Amine Functionalized Space-spanning Group)

A. Single Site

An oligonucleotide of the sequence:

Oligomer 5 GGC TGA* CTG CG SEQ ID NO:5

wherein A* indicates a 2'-O-(pentylamino) adenosine nucleotide was prepared as per the procedure of Example 1 utilizing an extend coupling time of 10 minutes during the coupling step of the modified nucleotide unit. 5'-Dimethoxytrityl-2'-O-(pentyl-N-phthalimido)-N6-benzyladenosine phosphoramidite (from Example 2-A-i was utilized in the DNA synthesizer as a 0.09M solution in anhydrous CH₃ CN. Oligonucleotide synthesis was carried out in either an ABI 390B or 394 synthesizer employing the standard synthesis cycles with an extended coupling time of 10 minutes during coupling of the 5'-dimethoxytrityl-2'-O-(pentyl-N-phthalimido)-N6-benzyladenosine phosphoramidite into the oligonucleotide sequence. Coupling efficiency of >98% was observed for the 5'-dimethoxytrityl-2'-O-(pentyl-N-phthalimido)-N6-benzyladenosine phosphoramidite coupling. Removal of the N-phthalimido amine protecting group is effected simultaneously with NH4OH deprotection of the benzyl and isobutyryl nucleotide base blocking groups.

B. Multiple Sites

An oligonucleotide of the sequence:

Oligomer 6 CTG TCT CCA* TCC TCT TCA* CT SEQ ID NO:6

wherein A* represents a nucleotide modified to incorporate a pentylamino functionality was prepared as per the procedures of Example 4-A. This oligonucleotide is an antisense compound to the E2 region of the bovine papilloma virus-1 (BPV-1).

EXAMPLE 5

A. Coupling of Oligonucleotide Strand Having An Abasic Site To An Oligonucleotide Having An Amine Functionalized Space-spanning Group

Two stranded, duplexed, cross-linked oligonucleotides of the structure:

Cross-linked Strands I ##STR1## where "|" represents an O--(CH₂)₅ --N--CH₂ -- cross-linkage between the oligonucleotide strands is prepared by reacting 2.8 OD. units of Oligomer 5, i.e. the GGC TGA* CTG CG oligonucleotide of Example 4-A, in 100 μl of 0.2M NaOAc buffer (pH 5.02) with 2.37 O.D. units of Oligomer 2, i.e. the abasic oligonucleotide CGC AGD* CAG CC of Example 3-A-iii, dissolved in 10 μl of water. The combined solution was let stand for 2 hours. 10 mg of NaCNBH₃ was dissolved in 400 μl of 250 mM NaOAc (pH 5.02) and 25 μl of this solution was added to the reaction solution. After this addition, the final concentration of the cyanoborohydride in the solution was nearly 80 mM. The reaction was followed at 1 hr intervals by HPLC. The amine linked oligonucleotide had a retention time of 21.65 mins and the abasic oligonucleotide 22.46 mins. A product peak was observed at 24.07 mins. A slow increase in the product peak was observed. After 12 hrs the product was separated from the reaction mixture by HPLC yielding 1.05 O.D. units, 20.3%. A small aliquot of the product was re-injected into the HPLC column and observed as a single peak.

B. Gel Analysis

A gel analysis was carried out on the above cross-linked 11 mer oligonucleotides, i.e. Cross-linked Strands I. The cross linked material moved slower than either of its component 11 mer oligonucleotides, i.e. Oligomers 2 or 5. Indeed it had the same electrophoretic mobility as that of a 21 mer oligonucleotide (11+11-1 for the loss of a base).

C. Ion Exchange Chromatography

Cross-linking of the Cross-linked Strands I was further confirmed by using ion exchange chromatography. The 21 mer cross-linked strands, since they have the greatest amount of charge, were eluted at 0.25M salt concentration whereas the 11 mer components oligonucleotides were eluted at 0.15M salt concentration.

D. Cross-Linking Affirmation

Cross-linking as opposed to hybridization duplex formation was confirmed independently by gel analysis and by re-injection of the cross-linked strands into HPLC. Gel analysis of a mixture the two component oligonucleotides mixed together without cross-linking did not yield the 21 mer mobility product. Re-injection of the cross-linked product into HPLC did not produce an equilibrium mixture of the two single oligonucleotide strands but came out as a single peak.

EXAMPLE 6

Preparation of Oligonucleotide Having Sulfur Linking Atom Attaching An Amine Functionalized Space-spanning Groups Thereon

An oligonucleotide of the sequence:

Oligomer 7 GGC T*GA CTG CG SEQ ID NO:7

wherein T* represents a nucleotide modified to incorporate a dodecylamino functionality joined to the thymidine nucleotide via a sulfur linking atom will be prepared as per the procedure of Example 4-A utilizing the oligonucleotide of Example 2-B-ii.

EXAMPLE 7

Preparation of Oligonucleotide Having Nitrogen Linking Atom Attaching An Amine Functionalized Space-spanning Groups Thereon

An oligonucleotide of the sequence:

Oligomer 8 GGC TGA* CTG CG SEQ ID NO:8

wherein A* represents a nucleotide modified to incorporate a butylamino functionality joined to the adenosine nucleotide via a nitrogen linking atom will be prepared as per the procedure of Example 4-A utilizing the oligonucleotide of Example 2-C-ii.

EXAMPLE 8

Preparation of Oligonucleotide Having Carbon Linking Atom Attaching An Amine Functionalized Space-spanning Groups Thereon

An oligonucleotide of the sequence:

Oligomer 9 GGC TGA* CTG CG SEQ ID NO:9

wherein A* represents a nucleotide modified to incorporate a allylamine functionality joined to the adenosine nucleotide via a methylene linking atom will be prepared as per the procedure of Example 4-A utilizing the oligonucleotide of Example 2-D-i.

EXAMPLE 9

A. Coupling of Oligonucleotide Strands Via Abasic Site and Amine Functionalized Space-spanning Group Connecting Through A Sulfur Linking Group

Two stranded, duplexed, cross-linked oligonucleotides of the structure:

Cross-linked Strands II ##STR2## where "|" indicates represents an S--(CH₂)₁₂ --N--CH₂ -- cross-linkage between the individual oligonucleotides of the structure will be prepared by reacting Oligomer 7 of Example 6 with the abasic Oligomer 2 of Example 3-A-iii utilizing the procedure of Example 5-A.

B. Coupling of Oligonucleotide Strands Via Abasic Site and Amine Functionalized Space-spanning Group Connecting Through A Nitrogen Linking Group

Two stranded, duplexed, cross-linked oligonucleotides of the structure:

Cross-linked Strands III ##STR3## where "|" indicates represents an --NH--(CH₂)₄ --N--CH₂ -- cross-linking between the individual oligonucleotides of the structure will be prepared by reacting Oligomer 8 of Example 7 with the abasic Oligomer 2 of Example 3-A-iii utilizing the procedure of Example 5-A.

C. Coupling of Oligonucleotide Strands Via Abasic Site and Amine Functionalized Space-spanning Group Connecting Through A Methylene Linking Group

Two stranded, duplexed, cross-linked oligonucleotides of the structure:

Cross-linked Strands IV ##STR4## where "|" indicates represents an --CH₂ --CH═CH--N--CH₂ -- cross-linkage between the individual oligonucleotides of the structure will be prepared by reacting Oligomer 9 of Example 8 with the abasic Oligomer 2 of Example 3-A-iii utilizing the procedure of Example 5-A.

EXAMPLE 10

A. Preparation of Oligonucleotide Having A Hydrazine Functionalized Space-spanning Groups Thereon

i. Single Functionality

An oligonucleotide of the sequence:

Oligomer 10 GGC TGU* CTG CG SEQ ID NO:10

wherein U* represents a nucleotide modified to incorporate a 2'-O-(octylhydrazino) functionality joined to the uridine nucleotide will be prepared as per the procedure of Example 4-A utilizing the oligonucleotide of Example 2-E-ii followed by deblocking of the benzylcarbazide protecting group via hydrogenolysis in the presence of palladium on charcoal.

ii. Multiple Functionalities

An oligonucleotide of the structure:

Oligomer 11 AGC CAG AU* C U GA GCC UGG G**AG CUC UCU GGC U SEQ ID NO:11

wherein U* represents an uridine nucleotide having a 2'-O-(octylhydrazine) group thereon and G** represents a guanosine nucleotide having a 2'-O-(octylhydrazine) group thereon will be prepared as per the procedure of Example 4-A utilizing the nucleotides of Examples 2-E-ii and 2-E-iv, respectively, to introduce the hydrazine functionalized, space-spanning group containing nucleotides in the proper sequence. Deblocking of the benzylcarbazide protecting group is effected via hydrogenolysis in the presence of palladium on charcoal.

B. Preparation of Oligonucleotide Having A Hydroxylamine Functionalized Space-spanning Groups Thereon

An oligonucleotide of the sequence:

Oligomer 12 GGC TGU* CTG CG SEQ ID NO:12

wherein U* represents a nucleotide modified to incorporate a octyl-hydroxylamine functionality joined to the cytidine nucleotide via an oxygen linking atom will be prepared as per the procedure of Example 4-A utilizing the oligonucleotide of Example 2-F-iii followed by treatment of the O-phthalimide group with methylhydrazine in DCM to generate the hydroxylamine functionality on the octyl space-spanning group.

C. Preparation of Oligonucleotide Having A Semicarbazide Functionalized Space-spanning Groups Thereon

An oligonucleotide of the sequence:

Oligomer 13 GGC TGU* CTG CG SEQ ID NO:13

wherein U* represents a nucleotide modified to incorporate a pentyl-N-semicarbazide functionality joined to the uridine nucleotide via an oxygen linking atom will be prepared as per the procedure of Example 4-A utilizing the oligonucleotide of Example 2-G-i.

D. Preparation of Oligonucleotide Having A Hydrazide Functionalized Space-spanning Groups Thereon

An oligonucleotide of the sequence:

Oligomer 14 GGC TGU* CTG CG SEQ ID NO:14

wherein U* represents a nucleotide modified to incorporate an ethyl hydrazide functionality joined to the uridine nucleotide via an oxygen linking atom will be prepared as per the procedure of Example 4-A utilizing the oligonucleotide of Example 2-H-iii.

E. Preparation of Oligonucleotide Having An Alcohol Functionalized Space-spanning Groups Thereon

An oligonucleotide of the sequence:

Oligomer 15 GGC TGU* CTG CG SEQ ID NO:15

wherein U* represents a nucleotide modified to incorporate a heptan-7-ol functionality joined to the uridine nucleotide will be prepared as per the procedure of Example 4-A utilizing the oligonucleotide of Example 2-I-iii.

EXAMPLE 11

A. Coupling of Oligonucleotide Strands Via Abasic Site and Hydrazine or Hydrazone Functionalized Space-spanning Group

i. Hydrazine Linked Strands

Two stranded, duplexed, cross-linked oligonucleotides of the structure:

Cross-linked Strands V ##STR5## where "|" indicates represents an O--(CH₂)₈ --NH--NH--CH₂ -- cross-linked between the individual oligonucleotides of the structure will be prepared by reacting Oligomer 10 of Example 10-A-i with the abasic Oligomer 2 of Example 3-A-iii followed by reduction of the resulting hydrazone linkage with NaCNBH₃ in NaOAc buffer as per the procedure of Example 5-A to form the hydrazine linkage

ii. Hydrazone Linked Strands

The cross-linker strands of Example 13-A-i having a hydrazone cross-linkage can be formed by omitting the NaCNBH₃ reduction step of Example 13-A-i.

B. Coupling of Oligonucleotide Strands Via Abasic Site and Hydroxylamine or Oxime Functionalized Space-spanning Group

i. Hydroxylamine Linked Strands

Two stranded, duplexed, cross-linked oligonucleotides of the structure:

Cross-linked Strands VI ##STR6## where "|" indicates represents an O--(CH₂)₈ --NH--O--CH₂ -- cross-linkage between the individual oligonucleotides of the structure will be prepared by reacting Oligomer 12 of Example 10-B with the abasic Oligomer 2 of Example 3-A-iii followed by reduction of the resulting oxime linkage with NaCNBH₃ in NaOAc buffer as per the procedure of Example 5-A to form the hydroxylamine.

ii. Oxime Linked Strands

The cross-linker strands of Example 13-B-i having an oxime cross-linkage can be formed by omitting the NaCNBH₃ reduction step of Example 13-A-i.

C. Coupling of Oligonucleotide Strands Via Abasic Site and Semicarbazide or Semicarbazone Functionalized Space-spanning Group

i. Semicarbazide Linked Strands

Two stranded, duplexed, cross-linked oligonucleotides of the structure:

Cross-linked Strands VII ##STR7## where "|" indicates represents an O--(CH₂)₈ --NH--NH--CH₂ -- cross-linkage between the individual oligonucleotides of the structure will be prepared by reacting Oligomer 13 of Example 10-C with the abasic Oligomer 2 of Example 3-A-iii followed by reduction of the resulting semicarbazone linkage with NaCNBH₃ in NaOAc buffer as per the procedure of Example 5-A to form the semicarbazide linkage.

ii. Semicarbazone Linked Strands

The cross-linker strands of Example 13-A-i having a semicarbazone cross-linkage can be formed by omitting the NaCNBH₃ reduction step of Example 13-A-i.

D. Coupling of Oligonucleotide Strands Via Hemiacetal Linkage Across An Abasic Site and An Alcohol Functionalized Space-spanning Group

Two stranded, duplexed, cross-linked oligonucleotides of the structure:

Cross-linked Strands VIII ##STR8## where "|" indicates represents an O--(CH₂)₇ --O--CH(OH)-- cross-linkage between the individual oligonucleotides of the structure will be prepared by reacting Oligomer 15 of Example 10-E with the abasic Oligomer 2 of Example 3-A-iii in the presence of trifluoroacetic acid utilizing a modification of the procedure of Knorre, et al., "Nucleotide and Oligonucleotide Derivatives As Enzyme and Nucleic Acid Targeted irreversible Inhibitors", Chemical Aspects, G. Weber (Ed.), Advance Press, Oxford, 1986, pp. 277-320.

EXAMPLE 12

Preparation of Oligonucleotide Having Multiple Space-spanning Groups That Include The Same Or Different Functionalities Thereon

A. Aldehyde and Amine Functionality Containing Oligonucleotide

A oligonucleotide of the structure:

Oligomer 16 AGC CAG AUC UGA* GCC UGG GAG CU**C UCU GGC U SEQ ID NO:16

wherein A* represents an adenosine nucleotide having a 2'-O-(pentylamino) group thereon and U** represents a uridine nucleotide having a 2'-O- propion-4-al bis(o-nitrobenzyl) acetal! group thereon will be prepared as per the procedure of Example 4-A utilizing the nucleotides of Examples 2-A-i and 2-K-i-b, respectively, for the functionalized space-spanning group containing nucleotides.

B. Multiple Thiol Functionality Containing Oligonucleotide

A oligonucleotide of the structure:

Oligomer 17 A*GC CAG AUC U GA GCC UGG GAG CUC UCU GGC** SEQ ID NO:17

wherein A* represents an adenosine nucleotide having a 2'-O-- S-trityl(hexyl-8-thiol)! group thereon and U** represents a uridine nucleotide having a 2'-O-- S-trityl(hexyl-8-thiol)! group thereon will be prepared as per the procedure of Example 4-A utilizing the nucleotides of Examples 2-J-ii-b and 2-J-ii-c, respectively, for the functionalized space-spanning group containing nucleotides.

C. Multiple Aldehydic Functionality Containing Oligonucleotide

An oligonucleotide of the structure:

Oligomer 18 GAC AGA GGU* AGG AGA AU* GA SEQ ID NO:18

wherein U* is a 2'-O- propion-3-al bis(o-nitrobenzyl) acetal! group will be prepared as per the procedure of Example 4-A utilizing the nucleotide of Example 2-K-i-b for the aldehydic functionalized space-spanning group containing nucleotides.

D. Thiol and Amine Functionality Containing Oligonucleotide

A oligonucleotide of the structure:

Oligomer 19 AGC CAG* AUC U GA GCC UGG GAG CUC U**CU GGC U SEQ ID NO:19

wherein G* represents an guanosine nucleotide having a 2'-O-( 2-amylenyl!-N-phthalimide) group thereon and U** represents a uridine nucleotide having a 2'-O- S-trityl(pentan-8-thiol)! group thereon will be prepared as per the procedure of Example 4-A utilizing the nucleotides of Examples 2-A-iv and 2-J-ii-e, respectively, for the functionalized space-spanning group containing nucleotides.

EXAMPLE 13

Multi-Site Cross-Linking Of Dual Strands of Oligonucleotides

A. Cross-Linking Via Dual Abasic Sites

Two stranded duplexed, multi-site cross-linked oligonucleotides of the structure:

Cross-linked Strand IX ##STR9## wherein "|" represents a cross-linkage between the strands, are prepared from Oligomer 4 from Example 3-A-iv and Oligomer 6 from Example 4-B utilizing the cross-linking procedure of Example 5-A. The resulting cross-linked strands are held together not only by hybridization of the duplex structures but also by the dual cross-linkages extending between the individual oligonucleotide strands.

B. Via Dual Space-spanning Groups

Two stranded duplexed, multi-site cross-linked oligonucleotides of the structure:

Cross-linked Strands X ##STR10## wherein "|" represents a cross-linkage having a covalent bond formed via an oxime linkage between the strands, is prepared from Oligomer 18 from Example 12-C and Oligomer 6 from Example 4-B. A solution of Oligomer 18 in water will be treated with hydrazine hydrate/ sodium cyanoborohydride to remove the aldehydic protecting groups.

Oligomer 6 is then added in 0.1M NaCl to hybridize the strands. Sodium acetate buffer is then added to effect cross-linking of the strands via formation of Schiff's bases between the amine and aldehyde groups. NaCNBH₃ is added to reduced the oxime linkage to an amine linkage whereby the resulting cross-linked strands will be held together not only by hybridization of the duplex structures but also by the multiple cross-links extending between the individual oligonucleotide strands.

EXAMPLE 14

Coupling of Two Sites on Single Strand

A single strand duplexed, cross-linked, hairpin loop oligonucleotide of the structure:

Cross-linked Strand XI ##STR11## wherein "|" represents a cross-linkage having a covalent bond formed via an oxime linkage between the regions of the strand, is prepared from Oligomer 16 from Example 12-A. Oligomer 16 is taken up in 0.1M NaCl solution to effect hybridization and then treated with hydrogen in the presence of palladium on charcoal at atmospheric pressure until two equivalents of hydrogen are taken up indicating removal of the aldehydic protecting groups. Sodium acetate buffer is then added to effect the formation of a Schiff's base linkage between the amine and aldehyde groups. NaCNBH₃ is added to reduced the oxime linkage to an amine linkage whereby the resulting cross-linked strand will be held together not only by hybridization of the duplex stem portion of the hairpin loop structure but also by the covalent cross-linkage extending across the stem nucleotides.

EXAMPLE 15

Coupling Via Disulfide Linkage

A single strand of duplexed, cross-linked, bulged, hairpin loop oligonucleotide of the structure:

Cross-linked Strand XII ##STR12## wherein "|" represents a cross-linkage having a covalent bond formed via a disulfide linkage between the regions of the strand, is prepared from Oligomer 17 from Example 12-B.

Oligomer 17 will be solubilized in 0.1M NaCl solution under nitrogen to effect hybridization and then treated with silver nitrate to remove the S-trityl protecting groups. A disulfide bond is formed between the two free thiol groups via mild oxidation by bubbling air through the solution to cross-linkage the A-U site of the oligonucleotide. The oligonucleotide will be held in the hairpin loop structure not only by hybridization of the duplexed stem portions of the hairpin loop structure but also by the covalent disulfide bond connecting the stem regions.

EXAMPLE 16

Coupling Via Dialdehyde

Utilizing the procedure of Example 4-A an oligonucleotide of the sequence:

Oligomer 20 A*GC CAG AUC U GA GCC UGG GAG CUC UCU GGC U SEQ ID NO:20

wherein A* is a 2'-O-(eicosylamino)adenosine nucleotide of Example 2-A-ii-c is prepared. The oligonucleotide is removed from the CPG synthetic column and de-salted on Sephadex G-25 Oligomer 20 will then be treated as per the peroxidation procedure of Lemairte et. al., Proc. Natl. Acad. Sci. USA, 84:648 (1986) to effect periodate-oxidation of the sugar moiety of the 3' terminus uridine nucleotide.

A single strand of duplexed, cross-linked, hairpin loop oligonucleotide of the structure:

Cross-linked Strand XIII ##STR13## wherein "|" represents a covalent cross-linkage formed via the oxidized, ring opened sugar of the 3'-terminal nucleotide and the amine group on the opposing nucleotide of the duplexed stem portion of the strand. Such cross-linkage is formed from the periodate-oxidized oligonucleotide by solubilizing the oxidized oligonucleotide in 0.1M NaCl to effect hybridization followed by the addition of sodium acetate buffer and NaCNBH₃. As the intermediate Schiff's base cross-linkage is formed, it is reduced to an amine linkage by the addition of NaCNBH₃ as per the procedure of Example 5-A.

EXAMPLE 17

Coupling Via Acetal Linkage

Utilizing the procedure of Example 4 an oligonucleotide, of, the sequence:

Oligomer 21 A*. GC CAG AVC V GA GCC VGG GAG CVC VCV GGC V SER. ID NO:21

wherein A* is a 2'-O- propion-3-al bis(o-nitrobenzyl) acetal! functionalized adenosine nucleotide will be prepared as per the procedure of Example 4-A utilizing the nucleotide of Example 2-K-i-a to introduce the aldehydic functionalized space-spanning group containing nucleotide with the oligonucleotide sequence.

The oligonucleotide is removed from the CPG synthetic column and de-salted on Sephadex G-25.

A single strand of duplexed, cross-linked, hairpin loop oligonucleotide of the structure:

Cross-linked Strand XIV ##STR14## wherein "|" represents an acetal cross-linkage formed between the 2',3'-hydroxyl moieties of the sugar on the 3'-terminal U nucleotide and the aldehydic functionality on the opposing A nucleotide of the duplexed stem portion of the strand, is prepared by effecting cross-linking in 0.1M NaCl in the presence of trifluoroacetic acid. This procedure is a modification of the procedure of Knorre et. al., "Nucleotide and Oligonucleotide Derivatives As Enzyme and Nucleic Acid Targeted Irreversible Inhibitors", Chemical Aspects, G. Weber (Ed.), Advance Press, Oxford, 1986, pp. 277-320. Upon reaction of the free aldehydic group on the A nucleotide, cross-linking to the opposing 2',3'-hydroxyl's of the 3'-terminus U nucleoside will yield a covalent cross-linkage between nucleotides of the stem portion of the hairpin loop bulged single stranded oligonucleotide.

EXAMPLE 18

Coupling Via Heterobifunctional Linker on Single Strand

A single strand of duplexed, cross-linked, hairpin loop oligonucleotide of the structure:

Cross-linked Strand XV ##STR15## wherein "\" represents a cross-linkage formed via a homobifunctional linker between the G and U nucleotides of duplexed regions of the strand across the G and U nucleotides of the two separate regions that are one base removed from Watson/Crick base pairs, is prepared from Oligomer 19 from Example 12-D. The Oligomer 19 will first be taken up in a 0.1M NaCl solution to effect hybridization of the oligonucleotide into a hairpin loop single strand duplexed structure. The reaction solution is then treated with silver nitrate in buffer to remove the S-trityl blocking group. A aliquot of N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) (Pierce, Rockford, Ill.) in weak alkaline is added to effect coupling of the amine functionality to the linker. The pH is then adjusted to pH 4.5 to effect exchange of the 2-pyridyl-disulfide and the thiol functionality on the oligonucleotide and in doing so to effect cross-linking of the strand. The strand will thus be held together not only by hybridization of the duplex stem portion of the hairpin loop bulged structure but also by the heterobifunctional cross-linker extending across the stem nucleotides.

EXAMPLE 19

Coupling Via Homobifunctional Linker

A single strand of duplexed, cross-linked, bulged, hairpin loop oligonucleotide of the structure:

Cross-linked Strand XVI ##STR16## wherein a cross-linkage is formed via a homobifunctional linker spanning between the U^(x) nucleotide on the UCU bulge and the G^(x) nucleotide of the AGGGUC loop regions of the strand, is prepared from Oligomer 11 from Example 10-A-ii. Molecular models of the proposed tertiary structure of Strand XVI indicate that the U^(x) and G^(x) nucleotides lie in close physical proximity to each other. To prepare Strand XVI the Oligomer 11 will first be taken up in a 0.1M NaCl solution to effect hybridization of the oligonucleotide into a hairpin loop bulged single strand duplexed structure. The reaction solution is then adjusted to be a 0.2M sodium borate buffer. Disuccinimidyl suberate (DSS, Pierce, Rockford, Ill.) is added to effect cross-linking of the hydrazine reactive functionalities of the uridine-guanosine nucleotides. The U nucleotide of the bulge and G nucleotide of the loop are linked via hydrazide linkages between the functionalities on the space-spanning groups and the homobifunctional cross-linker. In this structure, the U^(x) nucleotide of the interior loop and the G^(x) nucleotide of the hairpin loop are neither complementary to nor hybridizable with one another but they are located in 3-dimensional space in proximity with each other because of the higher order structure of the oligonucleotide. The cross-linkage spans this 3-dimensional space to fix the oligonucleotide in the higher order structure.

EXAMPLE 20

Synthesis of Uridine-2'-hexyl Aminolinker

Preparation of 5'-dimethoxytrityl-2'-(O-hexyl-N-phthalimido) uridine phosphoramidite

2',3'-O-Dibutyl stannylene-uridine was synthesized according to the procedure of Wagner, et al., J. Org. Chem., 1974, 39, 24. This compound was dried over P₂ O₅ under vacuum for 12 hrs. To a solution of this compound (29 g, 42.1 mmols) in 200 ml of anhydrous DMF were added (16.8 g, 50 mmols) of 6-bromohexyl phthalimide and 4.5 g of sodium iodide and the mixture was heated at 130° C. for 16 hrs. under argon. The reaction mixture was evaporated, co-evaporated once with toluene and the gummy tar residue was applied on a silica column (500 g). The column was washed with 2 L of EtOAc followed by eluting with 10% MeOH:90% EtOAc. The product, 2'- and 3'-isomers of O-hexyl-Ω-N-phthalimido uridine, eluted as an inseparable mixture (R_(f) =0.64 in 10% MeOH in EtOAc). By ¹³ C NMR, the isomeric ratio was ca. 55% of the 2' isomer and ca. 45% of the 3' isomer. The combined yield was 9.2 g (46.2%). This mixture was dried under vacuum and re-evaporated twice with pyridine. It was dissolved in 150 mL anhydrous pyridine and treated with 7.5 g of dimethyoxytrityl chloride (22.13 mmols) and 500 mg of DMAP. After 2 hrs. TLC(6:4 EtOAc:Hexane) indicated complete disappearance of the starting material and a good separation between 2' and 3' isomers (R_(f) =0.29 for the 2' isomer and 0.12 for the 3' isomer). The reaction mixture was quenched by the addition of 5 ML of CH₃ OH and evaporated under reduced pressure. The residue was dissolved in 300 mL CH₂ Cl₂, washed successively with saturate NaHCO3 followed by saturated NaCl solution. It was dried (MgSO₄) and evaporated to give 15 g of a brown foam which was purified on a silica gel (500 g) to give 6.5 g of the 2'-isomer and 3.5 g of the 3' isomer.

The 2'-O-hexyl-Ω-N-phthalimido-5'-DMT-uridine (4 g, 5.2 mmole) was dissolved in 40 ML of anhydrous CH₂ Cl₂. To this solution diisopropylaminetetrazolide (0.5 g, 2.9 mmol) and bis(isopropylamino)-β-cyanoethoxyphosphine (2.5 mL, 7.0 mmol) were added and stirred overnight. TLC (1:1 EtoAC/hexane) showed complete disappearance of starring material. The reaction mixture was transferred with CH₂ Cl₂ and washed with saturated NaHCO₃ (100 ML) followed by saturated NaCl solution. The organic layer was dried over anhydrous Na₂ SO₄ and evaporated to yield 6.4 g of a crude product which was purified in a silica column (200 g) using 1:1 hexane EtOAc to give 4.6 g (4.7 mmol, 90%) of the desired phosphoramidite.

EXAMPLE 21

Oligomer Synthesis and NMR characterization of an oligonucleotide containing U-2'-O-hexylamine linker

An oligonucleotide of the sequence:

Oligomer 22 TU*T SEQ ID NO:22

where U* represents the hexylamine modified uridine moiety, was synthesized on a 10 mmol synthesis in the DNA synthesizer. The phosphoramidite from Example 20 was used as a 0.12M solution in CH₃ CN. After deprotection of the oligonucleotide it was purified in a reverse phase HPLC column to get the pure trimer (any oligonucleotide containing undeprotected phthalimido group was separated from the trimer). The oligonucleotide was dried, desalted and dissolved in 600 uL D₂ O (app. 50 O.D., A₂₆₀). Both the proton and NMR ³¹ P NMR were examined. ¹ H NMR: 1.0-1.8 (m, CH2 protons from the hexyl chain); 5.75 (d); 5.9(d); 6.1 (t); 6.15 (t); 7.5 (s). ³¹ p NMR: -0.2; -0.4 ppm (two lines).

EXAMPLE 22

Synthesis illustrating preparation of any nucleotide (A, U, C or G) placed opposite to an abasic site in an opposing strand

A. Oligonucleotide Synthesis

An oligonucleotide of the sequence:

Oligomer 23 GGC TGU* CTG CG (P═O) SEQ ID NO:24

wherein U* indicates a 2'-O-(hexylamino) uridine nucleotide was prepared using an extended coupling time of 10 minutes. 5'-Dimethyoxytrityl-2'-O-(hexyl-N-phthalimido) uridine phosphoramidite was used as a 0.12M solution in anhydrous CH3CN. The oligonucleotide was deprotected, purified in reverse phase HPLC (trityl-on), deprotected at the 5'-position and purified again to remove any undeprotected phthalimido oligonucleotide.

B. Cross linking reaction

The Oligomer 23 oligonucleotide was reacted with the abasic oligonucleotide CGC AGD* CAG CC (Oligomer 2) with a crosslinking efficient of >90%. The formation of the conjugate was confirmed by reverse phase HPLC, ion-exchange HPLC and finally by the observation of base paired crosslinked duplex imino protons in the NMR spectrum.

EXAMPLE 23

Formation of a crosslink between a chimeric oligonucleotide and an abasic site containing oligonucleotide

An oligonucleotide of the sequence:

Oligomer 24 G_(s) G_(s) C_(o) T_(o) G_(o) U*_(o) C_(o) T_(o) G_(s) C_(s) G SEQ ID NO:24

where s represents a phosphorothioate backbone, o represents a phosphodiester backbone and U* represents a 2'-O-(hexylamino) uridine, was synthesized with a mixed (phosphodiester/phosphorothioate) backbone using either a conventional iodine oxidation (P═O) or a Beaucage reagent oxidation (P═S) in the respective oxidation steps. The oligonucleotide was synthesized, purified and crosslinked with CGCAGD*CAGCC. The crosslinked duplex was characterized by ¹ H NMR, Gel analysis, HPLC (reverse phase and ion-exchange).

EXAMPLE 24

Cross linked nucleic acid duplex involving connectivity between two abasic sites

Oligonucleotides with the following sequences were synthesized:

Oligomer 2 CGC AGD* CAG CC SEQ ID. NO:2

Oligomer 26 GGC TGD* CTG CG. SEQ ID NO:26

Oligomer 26 was obtained from a precursor oligomer:

Oligomer 25 GGC TGU CTG CG SEQ ID NO:25

where U represents 2'-deoxyuridine, by the action of uracil-DNA glycosylase. Oligomer 2 is reacted with 1,6-hexanediamine in NaOAc/NaCNBH₃ medium and a product isolated that has a free amino group located on the linker. This modified oligonucleotide is mixed with oligomer 26 in NaOAc buffer followed by the addition of NaCNBH3 in NaOAC buffer. The resultant cross link has a connectivity between two abasic sites separated by the --NH--(CH₂)₆ --NH-- linker. ##STR17## where "|" represents a --CH2--NH--(CH₂)₆ --NH--CH2crosslinker.

EXAMPLE 25

T_(m) determination of the Cross-linked Strands I.

The melting behavior of the crosslinked duplex, Cross-linked Strand I, was determined at 100 mM Nacl, 10 mM sodium phosphate, 0.1 mM EDTA at pH 7.0 at 4 mM each strand concentration.

    ______________________________________                                                                  T.sub.m                                               ______________________________________                                         Oligomer 5 - Oligomer 1 duplex (wild type)                                                                48° C.                                       Oligomer 5 - Oligomer 2 duplex (abasic site)                                                              <30° C.                                      Oligomer 5 - Oligomer 2 Cross-linked Strands I                                                            74° C.                                       ______________________________________                                    

The higher T_(m) of the crosslinked duplex confirmed the covalent linkage between the two strands (Oligomer 5 and Oligomer 2).

EVALUATION

PROCEDURE 1--Nuclease Resistance

A. Evaluation of the resistance of cross-linked oligonucleotides to serum and cytoplasmic nucleases.

Cross-linked oligonucleotides of the invention can be assessed for their resistance to serum nucleases by incubation of the cross-linked oligonucleotide in media containing various concentrations of fetal calf serum or adult human serum. Labeled cross-linked oligonucleotides are incubated for various times, treated with protease K and then analyzed by gel electrophoresis on 20% polyacrylamine-urea denaturing gels and subsequent autoradiography. Autoradiograms are quantitated by laser densitometry. Based upon the location of the modified linkage and the known length of the cross-linked oligonucleotide it is possible to determine the effect on nuclease degradation by the particular modification. For the cytoplasmic nucleases, an EL-60 cell line can be used. A post-mitochondrial supernatant is prepared by differential centrifugation and the labelled cross-linked oligonucleotides are incubated in this supernatant for various times. Following the incubation, the cross-linked oligonucleotides are assessed for degradation as outlined above for serum nucleolytic degradation. Autoradiography results are quantitated for comparison of the unmodified and the cross-linked oligonucleotides of the invention. It is expected that the cross-linked oligonucleotides will be completely resistant to serum and cytoplasmic nucleases.

B. Evaluation of the resistance of cross-linked oligonucleotides to specific endo- and exo-nucleases

Evaluation of the resistance of natural oligonucleotides and cross-linked oligonucleotides of the invention to specific nucleases (i.e., endonucleases, 3',5'-exo-, and 5',3'-exonucleases) can be done to determine the exact effect of the modified linkage on degradation. The cross-linked oligonucleotides are incubated in defined reaction buffers specific for various selected nucleases. Following treatment of the products with protease K, urea is added and analysis on 20% polyacrylamide gels containing urea is done. Gel products are visualized by staining with Stains All reagent (Sigma Chemical Co.). Laser densitometry is used to quantitate the extent of degradation. The effects of the modified linkage are determined for specific nucleases and compared with the results obtained from the serum and cytoplasmic systems. As with the serum and cytoplasmic nucleases, it is expected that the cross-linked oligonucleotides of the invention will be completely resistant to endo- and exo-nucleases.

PROCEDURE 2--5-Lipoxygenase Analysis and Assays

A. Therapeutics

For therapeutic use, an animal suspected of having a disease characterized by excessive or abnormal supply of 5-lipoxygenase is treated by administering cross-linked oligonucleotides in accordance with this invention. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Such treatment is generally continued until either a cure is effected or a diminution in the diseased state is achieved. Long term treatment is likely for some diseases.

B. Research Reagents

The cross-linked oligonucleotides of this invention will also be useful as research reagents when used to cleave or otherwise modulate 5-lipoxygenase mRNA in crude cell lysates or in partially purified or wholly purified RNA preparations. This application of the invention is accomplished, for example, by lysing cells by standard methods, optimally extracting the RNA and then treating it with a composition at concentrations ranging, for instance, from about 100 to about 500 ng per 10 μg of total RNA in a buffer consisting, for example, of 50 mm phosphate, pH ranging from about 4-10 at a temperature from about 30° to about 50° C. The cleaved 5-lipoxygenase RNA can be analyzed by agarose gel electrophoresis and hybridization with radiolabeled DNA probes or by other standard methods.

C. Diagnostics

The cross-linked oligonucleotides of this invention will also be useful in diagnostic applications, particularly for the determination of the expression of specific mRNA species in various tissues or the expression of abnormal or mutant RNA species. In this example, the cross-linked oligonucleotides target a hypothetical abnormal mRNA by being designed complementary to the abnormal sequence, but would not hybridize to normal mRNA.

Tissue samples can be homogenized, and RNA extracted by standard methods. The crude homogenate or extract can be treated for example to effect cleavage of the target RNA. The product can then be hybridized to a solid support which contains a bound oligonucleotide complementary to a region on the 5' side of the cleavage site. Both the normal and abnormal 5' region of the mRNA would bind to the solid support. The 3' region of the abnormal RNA, which is cleaved, would not be bound to the support and therefore would be separated from the normal mRNA.

Targeted mRNA species for modulation relates to 5-lipoxygenase; however, persons of ordinary skill in the art will appreciate that the present invention is not so limited and it is generally applicable. The inhibition or modulation of production of the enzyme 5-lipoxygenase is expected to have significant therapeutic benefits in the treatment of disease. In order to assess the effectiveness of the compositions, an assay or series of assays is required.

D. In Vitro Assays

The cellular assays for 5-lipoxygenase preferably use the human promyelocytic leukemia cell line HL-60. These cells can be induced to differentiate into either a monocyte-like cell or neutrophil-like cell by various known agents. Treatment of the cells with 1.3% dimethyl sulfoxide, DMSO, is known to promote differentiation of the cells into neutrophils. It has now been found that basal EL-60 cells do not synthesize detectable levels of 5-lipoxygenase protein or secrete leukotrienes (downstream products of 5-lipoxygenase). Differentiation of the cells with DMSO causes an appearance of 5-lipoxygenase protein and leukotriene biosynthesis 48 hours after addition of DMSO. Thus induction of 5-lipoxygenase protein synthesis can be utilized as a test system for analysis of cross-linked oligonucleotides which interfere with 5-lipoxygenase synthesis in these cells.

A second test system for cross-linked oligonucleotides makes use of the fact that 5-lipoxygenase is a "suicide" enzyme in that it inactivates itself upon reacting with substrate. Treatment of differentiated HL-60 or other cells expressing 5-lipoxygenase, with 10 μM A23187, a calcium ionophore, promotes translocation of 5-lipoxygenase from the cytosol to the membrane with subsequent activation of the enzyme. Following activation and several rounds of catalysis, the enzyme becomes catalytically inactive. Thus, treatment of the cells with calcium ionophore inactivates endogenous 5-lipoxygenase. It takes the cells approximately 24 hours to recover from A23187 treatment as measured by their ability to synthesize leukotriene B₄. Cross-linked oligonucleotides directed against 5-lipoxygenase can be tested for activity in two HL-60 model systems using the following quantitative assays. The assays are described from the most direct measurement of inhibition of 5-lipoxygenase protein synthesis in intact cells to more downstream events such as measurement of 5-lipoxygenase activity in intact cells.

The most direct effect which cross-linked oligonucleotides can exert on intact cells and which can be easily quantitated is specific inhibition of 5-lipoxygenase protein synthesis. To perform this technique, cells can be labelled with ³⁵ S-methionine (50 μCi/mL) for 2 hours at 37° C. to label newly synthesized protein. Cells are extracted to solubilize total cellular proteins and 5-lipoxygenase is immunoprecipitated with 5-lipoxygenase antibody followed by elution from protein A-Sepharose beads. The immunoprecipitated proteins are resolved by SDS-polyacrylamide gel electrophoresis and exposed for autoradiography. The amount of immunoprecipitated 5-lipoxygenase is quantitated by scanning densitometry.

A predicted result from these experiments would be as follows. The amount of 5-lipoxygenase protein immunoprecipitated from control cells would be normalized to 100%. Treatment of the cells with 1 μM, 10 μM, and 30 μM of effective cross-linked oligonucleotides for 48 hours would reduce immunoprecipitated 5-lipoxygenase by 5%, 25% and 75% of control, respectively.

Measurement of 5-lipoxygenase enzyme activity in cellular homogenates could also be used to quantitate the amount of enzyme present which is capable of synthesizing leukotrienes. A radiometric assay has now been developed for quantitating 5-lipoxygenase enzyme activity in cell homogenates using reverse phase HPLC. Cells are broken by sonication in a buffer containing protease inhibitors and EDTA. The cell homogenate is centrifuged at 10,000×g for 30 min and the supernatants analyzed for 5-lipoxygenase activity. Cytosolic proteins are incubated with 10 μM ¹⁴ C-arachidonic acid, 2 mM ATP, 50 μM free calcium, 100 μg/ml phosphatidylcholine, and 50 mM bis-Tris buffer, pH 7.0, for 5 min at 37° C. The reactions are quenched by the addition of an equal volume of acetone, and the fatty acids extracted with ethyl acetate. The substrate and reaction products are separated by reverse phase HPLC on a Novapak C18 column (Waters Inc., Millford, Mass.). Radioactive peaks are detected by a Beckman model 171 radiochromatography detector. The amount of arachidonic acid converted into di-HETE's and mono-HETE's is used as a measure of 5-lipoxygenase activity.

A predicted result for treatment of DMSO differentiated HL-60 cells for 72 hours with effective cross-linked oligonucleotides at 1 μM, 10 μM, and 30 μM would be as follows. Control cells oxidize 200 pmol arachidonic acid/5 min/10⁶ cells. Cells treated with 1 μM, 10 μM, and 30 μM of an effective cross-linked oligonucleotides would oxidize 195 pmol, 140 pmol, and 60 pmol of arachidonic acid/5 min/10⁶ cells respectively.

A quantitative competitive enzyme linked immunosorbant assay (ELISA) for the measurement of total 5-lipoxygenase protein in cells has been developed. Human 5-lipoxygenase expressed in E. coli and purified by extraction, Q-Sepharose, hydroxyapatite, and reverse phase HPLC is used as a standard and as the primary antigen to coat microtiter plates. 25 ng of purified 5-lipoxygenase is bound to the microtiter plates overnight at 4° C. The wells are blocked for 90 min with 5% goat serum diluted in 20 mM Tris-HCL buffer, pH 7.4, in the presence of 150 mM NaCl (TBS). Cell extracts (0.2% Triton X-100, 12,000×g for 30 min.) or purified 5-lipoxygenase were incubated with a 1:4000 dilution of 5-lipoxygenase polyclonal antibody in a total volume of 100 μL in the microtiter wells for 90 min. The antibodies are prepared by immunizing rabbits with purified human recombinant 5-lipoxygenase. The wells are washed with TBS containing 0.05% Tween 20 (TBST), then incubated with 100 μL of a 1:1000 dilution of peroxidase conjugated goat anti-rabbit IgG (Cappel Laboratories, Malvern, Pa.) for 60 min at 25° C. The wells are washed with TBST and the amount of peroxidase labelled second antibody determined by development with tetramethylbenzidine.

Predicted results from such an assay using a 30-mer cross-linked oligonucleotide at 1 μM, 10 μM, and 30 μM would be 30 ng, 18 ng and 5 ng of 5-lipoxygenase per 10⁶ cells, respectively, with untreated cells containing about 34 ng 5-lipoxygenase.

A net effect of inhibition of 5-lipoxygenase biosynthesis is a diminution in the quantities of leukotrienes released from stimulated cells. DMSO-differentiated HL-60 cells release leukotriene B4 (LTB4) upon stimulation with the calcium ionophore A23187. Leukotriene B4 released into the cell medium can be quantitated by radioimmunoassay using commercially available diagnostic kits (New England Nuclear, Boston, Mass.). Leukotriene B4 production can be detected in HL-60 cells 48 hours following addition of DMSO to differentiate the cells into neutrophil-like cells. Cells (2×10⁶ cells/mL) will be treated with increasing concentrations of cross-linked oligonucleotides for 48-72 hours in the presence of 1.3% DMSO. The cells are washed and resuspended at a concentration of 2×10⁶ cell/mL in Dulbecco's phosphate buffered saline containing 1% delipidated bovine serum albumin. Cells are stimulated with 10 μM calcium ionophore A23187 for 15 min and the quantity of LTB4 produced from 5×10⁵ cells determined by radioimmunoassay as described by the manufacturer.

Using this assay the following results would likely be obtained with a cross-linked oligonucleotide directed to the 5-lipoxygenase mRNA. Cells will be treated for 72 hours with either 1 μM, 10 μM or 30 μM cross-linked oligonucleotide in the presence of 1.3% DMSO. The quantity of LTB₄ produced from 5×10⁵ cells would be expected to be about 75 pg, 50 pg, and 35 pg, respectively with untreated differentiated cells producing 75 pg LTB₄.

E. In Vivo Assay

Inhibition of the production of 5-lipoxygenase in the mouse can be demonstrated in accordance with the following protocol. Topical application of arachidonic acid results in the rapid production of leukotriene B₄, leukotriene C₄ and prostaglandin E₂ in the skin followed by edema and cellular infiltration. Certain inhibitors of 5-lipoxygenase have been known to exhibit activity in this assay. For the assay, 2 mg of arachidonic acid is applied to a mouse ear with the contralateral ear serving as a control. The polymorphonuclear cell infiltrate is assayed by myeloperoxidase activity in homogenates taken from a biopsy 1 hour following the administration of arachidonic acid. The edematous response is quantitated by measurement of ear thickness and wet weight of a punch biopsy. Measurement of leukotriene B₄ produced in biopsy specimens is performed as a direct measurement of 5-lipoxygenase activity in the tissue. Cross-linked oligonucleotides will be applied topically to both ears 12 to 24 hours prior to administration of arachidonic acid to allow optimal activity of the compounds. Both ears are pretreated for 24 hours with either 0.1 μmol, 0.3 μmol, or 1.0 μmol of the oligonucleotide prior to challenge with arachidonic acid. Values are expressed as the mean for three animals per concentration. Inhibition of polymorphonuclear cell infiltration for 0.1 μmol, 0.3 μmol, and 1 μmol is expected to be about 10%, 75% and 92% of control activity, respectively. Inhibition of edema is expected to be about 3%, 58% and 90%, respectively while inhibition of leukotriene B₄ production would be expected to be about 15%, 79% and 99%, respectively.

    __________________________________________________________________________     SEQUENCE LISTING                                                               (1) GENERAL INFORMATION:                                                       (iii) NUMBER OF SEQUENCES: 26                                                  (2) INFORMATION FOR SEQ ID NO:1:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 11 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-site                                                    (B) LOCATION:6                                                                 (D) OTHER INFORMATION: 2'-deoxy uridine nucleotide                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                        CGCAGUCAGCC11                                                                  (2) INFORMATION FOR SEQ ID NO:2:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 11 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-site                                                    (B) LOCATION:6                                                                 (D) OTHER INFORMATION: abasic site                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                        CGCAGNCAGCC11                                                                  (2) INFORMATION FOR SEQ ID NO:3:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 20 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-site                                                    (B) LOCATION:9                                                                 (D) OTHER INFORMATION: 2'- deoxyuridine nucleotide                             (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-site                                                    (B) LOCATION:18                                                                (D) OTHER INFORMATION: 2'- deoxyuridine nucleotide                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                        GACAGAGGUAGGAGAAGUGA20                                                         (2) INFORMATION FOR SEQ ID NO:4:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 20 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-site                                                    (B) LOCATION:9                                                                 (D) OTHER INFORMATION: abasic site                                             (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-site                                                    (B) LOCATION:18                                                                (D) OTHER INFORMATION: abasic site                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                        GACAGAGGNAGGAGAAGNGA20                                                         (2) INFORMATION FOR SEQ ID NO:5:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 11 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-site                                                    (B) LOCATION:6                                                                 (D) OTHER INFORMATION: 2'-O- (pentylamino)adenosine                            nucleotide                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                        GGCTGACTGCG11                                                                  (2) INFORMATION FOR SEQ ID NO:6:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 20 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-site                                                    (B) LOCATION:9                                                                 (D) OTHER INFORMATION: nucleotide modified to                                  incorporate a pentylamino functionality                                        (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-site                                                    (B) LOCATION:18                                                                (D) OTHER INFORMATION: nucleotide modified to                                  incorporate a pentylamino functionality                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                        CTGTCTCCATCCTCTTCACT20                                                         (2) INFORMATION FOR SEQ ID NO:7:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 11 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-site                                                    (B) LOCATION:4                                                                 (D) OTHER INFORMATION: nucleotide modified to                                  incorporate a dodecylamino functionality                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                        GGCTGACTGCG11                                                                  (2) INFORMATION FOR SEQ ID NO:8:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 11 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-site                                                    (B) LOCATION:6                                                                 (D) OTHER INFORMATION: nucleotide modified to                                  incorporate a butylamino functionality                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                        GGCTGACTGCG11                                                                  (2) INFORMATION FOR SEQ ID NO:9:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 11 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-site                                                    (B) LOCATION:6                                                                 (D) OTHER INFORMATION: nucleotide modified to                                  incorporate a allylamine functionality                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                        GGCTGACTGCG11                                                                  (2) INFORMATION FOR SEQ ID NO:10:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 11 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-site                                                    (B) LOCATION:6                                                                 (D) OTHER INFORMATION: nucleotide modified to                                  incorporate a 2'-O-(octylhydrazino) functionality                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                       GGCTGUCTGCG11                                                                  (2) INFORMATION FOR SEQ ID NO:11:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 31 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-site                                                    (B) LOCATION:8                                                                 (D) OTHER INFORMATION: uridine nucleotide having a                             2'-O- (octylhydrazine) group                                                   (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-site                                                    (B) LOCATION:19                                                                (D) OTHER INFORMATION: guanosine nucleotide having                             a 2'-O- (octylhydrazine) group                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                       AGCCAGAUCUGAGCCUGGGAGCUCUCUGGCU31                                              (2) INFORMATION FOR SEQ ID NO:12:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 11 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-site                                                    (B) LOCATION:6                                                                 (D) OTHER INFORMATION: nucleotide modified to                                  incorporate a octyl-hydroxylamine functionality                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                       GGCTGUCTGCG11                                                                  (2) INFORMATION FOR SEQ ID NO:13:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 11 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-site                                                    (B) LOCATION:6                                                                 (D) OTHER INFORMATION: nucleotide modified to                                  incorporate a pentyl-N-semicarbazide                                           functionality                                                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                       GGCTGUCTGCG11                                                                  (2) INFORMATION FOR SEQ ID NO:14:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 11 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-site                                                    (B) LOCATION:6                                                                 (D) OTHER INFORMATION: nucleotide modified to                                  incorporate an ethyl hydrazide functionality                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                       GGCTGUCTGCG11                                                                  (2) INFORMATION FOR SEQ ID NO:15:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 11 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-site                                                    (B) LOCATION:6                                                                 (D) OTHER INFORMATION: nucleotide modified to                                  incorporate a heptan-7-ol functionality                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                       GGCTGUCTGCG11                                                                  (2) INFORMATION FOR SEQ ID NO:16:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 31 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-site                                                    (B) LOCATION:12                                                                (D) OTHER INFORMATION: adenosine nucleotide having                             a 2'-O- (pentylamino) group                                                    (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-site                                                    (B) LOCATION:23                                                                (D) OTHER INFORMATION: uridine nucleotide having a                             2'-O- propion-4-al bis(o-nitrobenzyl) acetal!                                  group                                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                       AGCCAGAUCUGAGCCUGGGAGCUCUCUGGCU31                                              (2) INFORMATION FOR SEQ ID NO:17:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 31 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-site                                                    (B) LOCATION:1                                                                 (D) OTHER INFORMATION: adenosine nucleotide having                             a 2'-O- S-trityl(hexyl-8-thiol)!group                                          (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-site                                                    (B) LOCATION:31                                                                (D) OTHER INFORMATION: uridine nucleotide having a                             2'-O- S- trityl(hexyl-8-thiol)!group                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                       AGCCAGAUCUGAGCCUGGGAGCUCUCUGGCU31                                              (2) INFORMATION FOR SEQ ID NO:18:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 19 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-site                                                    (B) LOCATION:9                                                                 (D) OTHER INFORMATION: 2'-O- propion-3-al bis(o-                               nitrobenzyl) acetal!group                                                      (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-site                                                    (B) LOCATION:17                                                                (D) OTHER INFORMATION: 2'-O- propion-3-al bis(o-                               nitrobenzyl) acetal!group                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                       GACAGAGGUAGGAGAAUGA19                                                          (2) INFORMATION FOR SEQ ID NO:19:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 31 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-site                                                    (B) LOCATION:6                                                                 (D) OTHER INFORMATION: guanosine nucleotide having                             a 2'-O-( 2-amylenyl!-N-phthalimide) group                                      (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-site                                                    (B) LOCATION:25                                                                (D) OTHER INFORMATION: uridine nucleotide having a                             2'-O- S- trityl(pentan-8-thiol)!group                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                       AGCCAGAUCUGAGCCUGGGAGCUCUCUGGCU31                                              (2) INFORMATION FOR SEQ ID NO:20:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 31 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-site                                                    (B) LOCATION:1                                                                 (D) OTHER INFORMATION: 2'-O- (eicosylamino)adenosine                           nucleotide                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                       AGCCAGAUCUGAGCCUGGGAGCUCUCUGGCU31                                              (2) INFORMATION FOR SEQ ID NO:21:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 31 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-site                                                    (B) LOCATION:1                                                                 (D) OTHER INFORMATION: 2'-O- propion-3-al bis(o-                               nitrobenzyl) acetal!functionalized adenosine                                   nucleotide                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                       AGCCAGAUCUGAGCCUGGGAGCUCUCUGGCU31                                              (2) INFORMATION FOR SEQ ID NO:22:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 3 base pairs                                                       (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-site                                                    (B) LOCATION:2                                                                 (D) OTHER INFORMATION: hexylamine modified uridine                             moiety                                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                       TUT3                                                                           (2) INFORMATION FOR SEQ ID NO:23:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 11 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-site                                                    (B) LOCATION:6                                                                 (D) OTHER INFORMATION: 2'-O- (hexylamino) uridine                              nucleotide                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                       GGCTGUCTGCG11                                                                  (2) INFORMATION FOR SEQ ID NO:24:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 11 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-site                                                    (B) LOCATION:6                                                                 (D) OTHER INFORMATION: 2'-O- (hexylamino) uridine                              nucleotide                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                       GGCTGUCTGCG11                                                                  (2) INFORMATION FOR SEQ ID NO:25:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 11 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-site                                                    (B) LOCATION:6                                                                 (D) OTHER INFORMATION: 2'- deoxyuridine                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                       GGCTGUCTGCG11                                                                  (2) INFORMATION FOR SEQ ID NO:26:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 11 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-site                                                    (B) LOCATION:6                                                                 (D) OTHER INFORMATION: abasic site                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                       GGCTGNCTGCG11                                                                  __________________________________________________________________________ 

What is claimed is:
 1. A cross-linked nucleic acid comprising:a first nucleotide located on a first oligonucleo- tide strand; a first bond means located on either a 2'-hydroxyl or a 3'-hydroxyl of a sugar moiety of said first nucleotide; a second nucleotide located on a second oligonucleotide strand; a second bond means located on either a 2'-hydroxyl or a 3'-hydroxyl of a sugar moiety of said second nucleotide; and a non-phosphorous covalent cross-linkage between said first and said second bond means;wherein: said first bond means is located on a 2'-hydroxyl of a sugar moiety of said first nucleotide and said second bond means is located on a 3'-hydroxyl of a sugar moiety of said second nucleotide; or said first bond means is located on a 2'-hydroxyl of a sugar moiety of said first nucleotide and said second bond means is located on a 2'-hydroxyl of a sugar moiety of said second nucleotide; or said first bond means is located on a 3'-hydroxyl of a sugar moiety of said first nucleotide and said second bond means is located on a 2'-hydroxyl of a sugar moiety of said second nucleotide.
 2. The cross-linked nucleic acid of claim 1 wherein:said first nucleotide is located in a first nucleotide sequence and said second nucleotide is located in a second nucleotide sequence; and said second nucleotide sequence is complementary to and hybridizable with said first nucleotide sequence.
 3. The cross-linked nucleic acid of claim 1 wherein said first bond means is located on a 2'-hydroxyl of a sugar moiety of said first nucleotide and said second bond means is located on a 3'-hydroxyl of a sugar moiety of said second nucleotide.
 4. The cross-linked nucleic acid of claim 1 wherein said first bond means is located on a 2'-hydroxyl of a sugar moiety of said first nucleotide and said second bond means is located on a 2'-hydroxyl of a sugar moiety of said second nucleotide.
 5. The cross-linked nucleic acid of claim 1 wherein said first bond means is located on a 3'-hydroxyl of a sugar moiety of said first nucleotide and said second bond means is located on a 2'-hydroxyl of a sugar moiety of said second nucleotide.
 6. The cross-linked nucleic acid of claim 1 wherein said cross-linkage is --N═CH--, --N(R)--, --N(R)--CH₂ --NH--, --NH--CH₂ --NH--, --O--N═CH--, --O--N(R)--, --N(R)--N(R)--, --HC═N--N(R)--, --H═N--N═CH--, --C(O)--N(R)--N═CH--, --C(O)N(R)--, --C(O)--N(R)--N(R)--, --N(R)--C(O)--N(R)--N(R)--, --N(R)--C(O)--N(R)--N═CH--, --N(R)--C(S)--N(R)--NH--, --N(R)--C(S)--N(R)--N═CH--, --S--S--, --CH(OH)--O--CH₂ --, --CH(OH)--S--CH₂ --, --CH₂ --S--CH₂, C(O)--CH₂ -- or --CH₂ --NH--CH₂ --C(O)--CH₂ --, wherein R is H, alkyl, alkenyl, aralkyl, an intercalator, a conjugate, polyamine, polyamide, or polyethylene glycol.
 7. A cross-linked nucleic acid comprising:a first nucleotide located on an oligonucleotide strand; a first bond means located on either a 2'-hydroxyl or a 3'-hydroxyl of a sugar moiety of said first nucleotide; a second nucleotide located on said oligonucleotide strand; a second bond means located on either a 2'-hydroxyl or a 3'-hydroxyl of a sugar moiety of said second nucleotide; and a non-phosphorous covalent cross-linkage between said first and said second bond means.
 8. The cross-linked nucleic acid of claim 7 wherein:said first nucleotide is located in a first nucleotide sequence and said second nucleotide is located in a second nucleotide sequence; and said second nucleotide sequence is complementary to and hybridizable with said first nucleotide sequence.
 9. The cross-linked nucleic acid of claim 7 wherein said first bond means includes an abasic site.
 10. The cross-linked nucleic acid of claim 7 wherein said abasic site includes an aldehyde.
 11. The cross-linked nucleic acid of claim 7 wherein said first bond means is located on a 2'-hydroxyl of a sugar moiety of said first nucleotide and said second bond means is located on a 3'-hydroxyl of a sugar moiety of said second nucleotide.
 12. The cross-linked nucleic acid of claim 7 wherein said first bond means is located on a 2'-hydroxyl of a sugar moiety of said first nucleotide and said second bond means is located on a 2'-hydroxyl of a sugar moiety of said second nucleotide.
 13. The cross-linked nucleic acid of claim 7 wherein said first bond means is located on a 3'-hydroxyl of a sugar moiety of said first nucleotide and said second bond means is located on a 2'-hydroxyl of a sugar moiety of said second nucleotide.
 14. The cross-linked nucleic acid of claim 7 wherein said first bond means is located on a 3'-hydroxyl of a sugar moiety of said first nucleotide and said second bond means is located on a 3'-hydroxyl of a sugar moiety of said second nucleotide.
 15. The cross-linked nucleic acid of claim 7 wherein said cross-linkage is --N═CH--, --N(R)--, --N(R)--CH₂ --NH--, --NH--CH₂ --NH--, --O--N═CH--, --O--N(R)--, --N(R)--N(R)--, --HC═N--N(R)--, --HC═N--N═CH--, --C(O)--N(R)--N═CH--, --C(O)N(R)--, --C(O)--N(R)--N(R)--, --N(R)--C(O)--N(R)--N(R)--, --N(R)--C(O)--N(R)--N═CH--, --N(R)--C(S)--N(R)--NH--, --N(R)--C(S)--N(R)--N═CH--, --S--S--, --CH(OH)--O--CH₂ --, --CH(OH)--S--CH₂ --, --CH₂ --S--CH₂ --, C(O)--CH₂ -- or --CH₂ --NH--CH₂ --C(O)--CH₂ --, wherein R is H, alkyl, alkenyl, aralkyl, an intercalator, a conjugate, polyamine, polyamide, or polyethylene glycol.
 16. A cross-linked nucleic acid comprising:a first nucleotide located on a first oligonucleotide strand; a first bond means located on either a 2'-hydroxyl or a 3'-hydroxyl of a sugar moiety of said first nucleotide; a second nucleotide located on a second oligonucleotide strand; a second bond means located on either a 2'-hydroxyl or a 3'-hydroxyl of a sugar moiety of said second nucleotide; and a non-phosphorous covalent cross-linkage between said first and said second bond means, provided that at least one of said first nucleotide and said second nucleotide is not a terminal nucleotide.
 17. The cross-linked nucleic acid of claim 16 wherein:said first nucleotide is located in a first nucleotide sequence and said second nucleotide is located in a second nucleotide sequence; and said second nucleotide sequence is complementary to and hybridizable with said first nucleotide sequence.
 18. The cross-linked nucleic acid of claim 16 wherein said first bond means is located on a 2'-hydroxyl of a sugar moiety of said first nucleotide and said second bond means is located on a 3'-hydroxyl of a sugar moiety of said second nucleotide.
 19. The cross-linked nucleic acid of claim 16 wherein said first bond means is located on a 2'-hydroxyl of a sugar moiety of said first nucleotide and said second bond means is located on a 2'-hydroxyl of a sugar moiety; of said second nucleotide.
 20. The cross-linked nucleic acid of claim 16 wherein said first bond means is located on a 3'-hydroxyl of a sugar moiety of said first nucleotide and said second bond means is located on a 2'-hydroxyl of a sugar moiety of said second nucleotide.
 21. The cross-linked nucleic acid of claim 16 wherein said first bond means is located on a 3'-hydroxyl of a sugar moiety of said first nucleotide and said second bond means is located on a 3'-hydroxyl of a sugar moiety of said second nucleotide.
 22. The cross-linked nucleic acid of claim 16 wherein said cross-linkage is --N═CH₂ --, --N(R)--, --N(R)--CH₂ --NH--, --NH--CH₂ --NH--, --O--N═CH--, --O--N(R)--, --N(R)--N(R)--, --HC═N--N(R)--, --CH═N--N═CH--, --C(O)--N(R)--N═CH--, --C(O)N(R)--, --C(O)--N(R)--N(R)--, --N(R)--C(O)--N(R)--N(R)--, --N(R)--C(O)--N(R)--N═CH--, --N(R)--C(S)--N(R)--NH--, --N(R)--C(S)--N(R)--N═CH--, --S--S--, --CH(OH)--O--CH₂ --, --CH(OH)--S--CH₂ --, --CH₂ --S--CH₂ --, C(O)--CH₂ -- or --CH₂ --NH--CH₂ --C(O)--CH₂ --, wherein R is H, alkyl, alkenyl, aralkyl, an intercalator, a conjugate, polyamine, polyamide, or polyethylene glycol. 